Camptothecin analogs and methods of preparation thereof

ABSTRACT

A compound having the formula 
     
       
         
         
             
             
         
       
     
     in racemic form, enantiomerically enriched form or enantiomerically pure form, and pharmaceutically acceptable salts thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is divisional patent application of U.S. patentapplication Ser. No. 12/425,364, filed Apr. 16, 2009, which is adivisional patent application of U.S. patent application Ser. No.11/805,169, filed May 22, 2007, now U.S. Pat. No. 7,538,220, which is adivisional application of U.S. patent application Ser. No. 10/919,068,filed Aug. 16, 2004, now U.S. Pat. No. 7,220,860, which is a divisionalapplication of U.S. patent application Ser. No. 10/164,326, filed Jun.6, 2002, now U.S. Pat. No. 6,809,103, which is a divisional of U.S.patent application Ser. No. 09/728,031, now U.S. Pat. No. 6,410,731,filed Nov. 30, 2000, which is a continuation application of U.S. patentapplication Ser. No. 09/290,019, now U.S. Pat. No. 6,207,832, filed Apr.9, 1999, the disclosures of which are incorporated herein by reference.

GOVERNMENT INTERESTS

This invention was made with government support under grant number RO1GM031678 awarded by the National Institutes of Health. The governmenthas certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to novel compounds and methods ofpreparation thereof and, particularly, to E-ring expanded camptothecinderivatives or analogs and to methods of preparation of suchcamptothecin analogs.

BACKGROUND OF THE INVENTION

Camptothecins are DNA topoisomerase I inhibitors now being used asanticancer drugs. Topotecan (tpt) and CPT-11 are the first two membersin the camptothecin family to gain Food and Drug Administration fullapproval status (topotecan in 1996 as second-line therapy for advancedepithelial ovarian cancer, topotecan again in 1998 for the treatment ofsmall cell lung cancer, CPT-11 in 1998 as first-line therapy for coloncancer). Several other analogs of the camptothecin family such asGI-147211C, DX8951f, 9-aminocamptothecin (9-AC) and 9-nitrocamptothecinare in various stages of pre-clinical and clinical evaluation. Each ofthe campothecins in clinical use undergoes relatively rapid hydrolysisin the bloodsteam resulting in a marked loss of anticancer activity. Itis the key α-hydroxylactone pharmacophore within clinically relevantcamptothecins that hydrolyzes at physiological pH to yield abiologically-inactive and potentially toxic hydroxy carboxylate form.Fassberg, J. and Stella, V. J., “A Kinetic and Mechanistic Study of theHydrolysis of Camptothecin and Some Analogues”, J. Pharm. Sci. 81:676-684 (1992); Hertzberg, R. P., Caranfa, M. J., and Hecht, S. M., “Onthe Mechanism of Topoisomerase I Inhibition by Camptothecin: Evidencefor Binding to an Enzyme-DNA Complex”, Biochemistry 28: 4629-4638(1989); Hsiang, Y-H., and Liu, L. F., “Identification of Mammalian DNATopoisomerase I as an Intracellular Target of the Anticancer DrugCamptothecin”, Cancer Res. 48: 1722-1726 (1988); and Jaxel, C., Kohn, K.W., Wani, M. C., Wall, M. E., and Pommier, Y., “Structure-Activity Studyof Camptothecin Derivatives on Mammalian Topoisomerase I: Evidence for aSpecific Receptor Site and a Relation to Antitumor Activity”, CancerRes. 49: 5077-5082 (1989). References set forth herein, including thoseset forth above, may facilitate understanding of the present invention.Inclusion of a reference herein is not intended to an does no constitutean admission that the reference is prior art with respect to the presentinvention.

The structures of camptothecin and some of its important analogs areshown below:

Recent research efforts have shown that agents such as9-aminocamptothecin and camptothecin (cpt) display very poor stabilitiesin human blood due to high affinity binding interactions between theircarboxylate forms and human serum albumin (HSA). Burke, T. G, Mi, Z.,Jiang, Y., and Munshi, C. B. “The Important Role of Albumin inDetermining the Relative Human Blood Stabilities of the CamptothecinAnticancer Drugs”, Journal of Pharmaceutical Sciences, 84: 518-519(1995); Burke, T. G. and Mi, Z. “The Structural Basis of CamptothecinInteractions with Human Serum Albumin: Impact on Drug Stability”,Journal of Medicinal Chemistry, 37: 40-(1994); Mi, Z. and Burke, T. G.,“Differential interactions of Camptothecin Lactone and Carboxylate Formswith Human Blood Components”, Biochemistry, 33: 10325-10336 (1994); andMi, Z., Malak, H., and Burke, T. G. “Reduced Albumin Binding Promotesthe Stability and Activity of Topotecan in Human Blood”, Biochemistry,34: 13722-13728 (1995), the disclosures of which are incorporated hereinby reference. Frequency-domain lifetime fluorometry experiments revealedthat human serum albumin (HSA) preferentially binds camptothecincarboxylate with over a 100-fold higher affinity compared tocamptothecin lactone. Mi, Z. and Burke, T. G. “Marked InterspeciesVariations Concerning the Interactions of Camptothecin with SerumAlbumins: A Frequency-Domain Fluorescence Spectroscopic Study”,Biochemistry, 33: 12540-12545 (1994), the disclosure of which isincorporated herein by reference. This differential binding ofcarboxylate over lactone results in camptothecin and 9-AC opening morerapidly and completely in the presence of HSA than in the absence of theprotein. In human plasma, pH 7.4 and 37° C., camptothecin and 9-AC bothopen rapidly and essentially completely to almost negligible 0.2%lactone levels at equilibrium. While the presence of HSA promoteslactone ring opening for camptothecin and 9-AC, red blood cells andlipid bilayers in general preferentially bind the electroneutral lactoneforms of camptothecins over their respective negatively-chargedcarboxylate lactone forms. Burke, T. G., Staubus, A. E., Mishra, A. K.,and Malak, H., “Liposomal Stabilization of Camptothecin's Lactone Ring”,J. Am. Chem. Soc. 114: 8318-8319 (1992); and Burke, T. G., Mishra, A.K., Wani, M., and Wall, M., “Lipid Bilayer Partitioning and Stability ofCamptothecin Drugs”, Biochemistry, 32: 5352-5364 (1993), the disclosuresof which are incorporated herein by reference. Drug interactions witherythrocytes thereby promote active lactone levels in blood.

Recently, Layergne et al. have shown that expansion of the E-ring ofcamptothecin to produce a “homocamptothecin” enhances the solutionstability of camptothecin while maintaining anticancer activity.Layergne, O., Lesueur-Ginot, L., Rodas, F. P., Kasprzyk, P. G., Pommier,J., Demarquay, D., Prevost, G., Ulibarri, G., Rolland, A.,Schiano-Liberatore, A.-M., Harnett, J., Pons, D., Camara, J., Bigg, D.,“Homocamptothecins: Synthesis and Antitumor Activity of Novel E-RingModified Camptothecin Analogs”, J. Med. Chem., 41, 5410-5419 (1998); andLayergne, O., Lesueur-Ginot, L., Rodas, F. P., and Bigg, D., “An E-RingModified Camptothecin With Potent Antiproliferative and Topoisomerase Iinhibitory Activities. Bioorg. Med. Chem. Lett. 7, 2235-2238 (1997). Themodification to the E-ring in the studies of Layergne et al. involvedinsertion of a methylene spacer between the 20-OH functionality and thecarboxyl group of the naturally occurring six-membered α-hydroxylactoneof camptothecin. Incorporation of the new 7-membered β-hydroxylactonering into camptothecin was found to improve the solution and plasmastability of the agent.

The structure of the homocamptothecin of Layergne et al. and thenumbering system used to describe such compounds are shown below:

Although substantial strides have been made in the development of thecamptothecin family of drugs, it remains very desirable to developimproved compounds in this family of drugs and to develop improvedsynthetic routes for producing such drugs.

SUMMARY OF THE INVENTION

The present invention provides generally for a compound having thefollowing formula (1):

in racemic form, enantiomerically enriched form or enantiomerically pureform;wherein R¹ and R² are independently the same or different and arehydrogen, —C(O)R^(f) wherein R^(f) is an alkyl group, an alkoxy group,an amino group or a hydroxy group, an alkyl group, an alkenyl group, analkynyl group, an alkoxy group, an aryloxy group, an acyloxy group,—OC(O)OR^(d), wherein R^(d) is an alkyl group, —OC(O)NR^(a)R^(b) whereinR^(a) and R^(b) are independently the same or different, H, —C(O)R^(f),an alkyl group or an aryl group, a halogen, a hydroxy group, a nitrogroup, a cyano group, an azido group, a formyl group, a hydrazino group,an amino group, —SR^(c), wherein R^(c) is hydrogen, —C(O)R^(f), an alkylgroup or an aryl group; or R¹ and R² together form a chain of three orfour members selected from the group of CH, CH₂, O, S, NH, or NR¹⁵,wherein R¹⁵ is an C₁-C₆ alkyl group;R³ is H, a halogen atom, a nitro group, an amino group, a hydroxy group,or a cyano group; or R² and R³ together form a chain of three or fourmembers selected from the group of CH, CH₂, O, S, NH, or NR¹⁵, whereinR¹⁵ is an C₁-C₆ alkyl group;R⁴ is H, F, an amino group, a C₁₋₃ alkyl group, a C₂₋₃ alkenyl group, aC₂₋₃ alkynyl group, a trialkylsilyl group or a C₁₋₃ alkoxy group;R⁵ is a C₁₋₁₀ alkyl group, an alkenyl group, an alkynyl group, or abenzyl group;R⁶ is —Si(R⁸R⁹R¹⁰) or —(R⁷)Si(R⁸R⁹R¹⁰) wherein R⁷ is an alkylene group,an alkenylene group, or an alkynylene group; and R⁸, R⁹ and R¹⁰ areindependently a C₁₋₁₀ alkyl group, a C₂₋₁₀ alkenyl group, a C₂₋₁₀alkynyl group, an aryl group or a —(CH₂)_(N)R¹¹ group, wherein N is aninteger within the range of 1 through 10 and R¹¹ is a hydroxy group, analkoxy group, an amino group, an alkylamino group, a dialkylamino group,a halogen atom, a cyano group, —SR^(c) or a nitro group;

R¹³ is H, F or —CH₃; R¹⁶ is —C(O)R^(f) or H; and

pharmaceutically acceptable salts thereof.

R¹ and R² together may, for example, form a group of the formula—O(CH₂)_(n)O— wherein n represents the integer 1 or 2. Likewise, R² andR³ together may, for example, form a group of the formula —O(CH₂)_(n)O—wherein n represents the integer 1 or 2.

R⁵ is preferably an ethyl group, an allyl group, a benzyl group or apropargyl group. Most preferably, R⁵ is an ethyl group. Preferably, R⁴is H.

In one embodiment, R⁸ and R⁹ are methyl groups, R¹⁰ is a tert-butylgroup or a methyl group, R¹ is H and R³ is H. In this embodiment, R²may, for example, be H, NH₂ or OH.

R¹³ is preferably H. R¹⁶ is preferably H or an alkyl group. Mostpreferably, R¹⁶ is H or —C(O)R^(f), wherein R^(f) is an alkyl group.Most preferably, R¹⁶ is H.

The present invention also provides a method of synthesizing a compoundhaving the formula

via a cascade radical 4+1 annulation wherein the precursor

or the precursor

is reacted with an arylisonitrile having the formula

wherein X is a radical precursor. Preferably, X is Cl, Br or I. Mostpreferably, X is Br or I.

The present invention also provides a compound having the formula

in racemic form, enantiomerically enriched form or enantiomerically pureform, wherein R¹² is preferably H or —C(O)R^(f), —C(O)OR^(d) or—C(O)NR^(a)R^(b); andpharmaceutically acceptable salts thereof.

The present invention further provides compounds having the formulas

in racemic form, enantiomerically enriched form or enantiomerically pureform;

Still further, the present invention provides a compound having theformula

in racemic form, enantiomerically enriched form or enantiomerically pureform

The present invention also provides a compound having the formula

The present invention also provides a compound having the formula

wherein R¹⁵ is a C₁-C₆ alkyl group.

The present invention further provides a compound having the formula

in racemic form, enantiomerically enriched form or enantiomerically pureform, wherein R¹⁴ is SiMe₃, I, or Br.

Still further, the present invention provides a method of synthesizing acompound having the following formula:

wherein Y is chlorine, bromine or iodine;comprising the steps of(a) treating an enol ether of the structure:

under suitable oxidative cleavage conditions to form a compound havingthe structure:

(b) treating the compound formed in step (a) with an organometallicreagent having the structure:

MC(R¹³)(R¹³)CO₂R¹⁵

wherein M is Li, Na, K, MgY, or ZnY under suitable conditions to form acompound having the structure:

(c) treating the compound formed in step (b) under suitable conditionswith acid to form a compound having the structure:

(d) treating the compound formed in step (c) under suitable conditionsof halogenative desilylation to form a compound having the structure:

(e) treating the compound in step (d) with acid or iodotrimethylsilaneunder suitable conditions for demethylation to provide a compound of thefollowing structure:

(f) treating the compound in step (e) with a lithium base or a sodiumbase in the presence of an inorganic lithium salt to deprotonate thenitrogen atom,(g) reacting of the resulting deprotonated species of step (f) with acompound of the following structure:

wherein Z is I, Br, Cl, a mesylate group or a tosylate group, and undersuitable conditions to cause the formation of the compound of thefollowing structure:

As indicated above, all compounds of the present invention including theβ-hydroxylactone group can exist in racemic form, enantiomericallyenriched form, or enantiomerically pure form. The formulas of suchcompounds as set forth herein cover and/or include each such form.

The term “radical precursor(s)” as used herein and as well known tothose skilled in the art refers generally to those functional groupsthat cleave to generate radicals under standard conditions of chain ornon-chain radical reactions. Common radical precursors are the halogens(except fluorine), carboxylic acids and derivatives thereof (such asthiohydroxamates), selenophenyl groups, diazonium salts, and the like.See, for example, Giese, B. Radicals in Organic Synthesis: Formation ofCarbon-Carbon Bonds; Pergamon, Oxford (1986), the disclosure of which isincorporated herein by reference.

The terms “alkyl”, “aryl” and other groups refer generally to bothunsubstituted and substituted groups unless specified to the contrary.Unless otherwise specified, alkyl groups are hydrocarbon groups and arepreferably C₁-C₁₅ (that is, having 1 to 15 carbon atoms) alkyl groups,and more preferably C₁-C₁₀ alkyl groups, and can be branched orunbranched, acyclic or cyclic. The above definition of an alkyl groupand other definitions apply also when the group is a substituent onanother group (for example, an alkyl group as a substituent of analkylamino group or a dialkylamino group). The term “aryl” refers tophenyl or naphthyl. As used herein, the terms “halogen” or “halo” referto fluoro, chloro, bromo and iodo.

The term “alkoxy” refers to —OR^(d), wherein R^(d) is an alkyl group.The term “aryloxy” refers to —OR^(e), wherein R^(e) is an aryl group.The term acyl refers to —C(O)R^(f). The term “alkenyl” refers to astraight or branched chain hydrocarbon group with at least one doublebond, preferably with 2-15 carbon atoms, and more preferably with 2-10carbon atoms (for example, —CH═CHR^(g) or —CH₂CH═CHR^(g)). The term“alkynyl” refers to a straight or branched chain hydrocarbon group withat least one triple bond, preferably with 2-15 carbon atoms, and morepreferably with 2-10 carbon atoms (for example, —C≡CR^(h) or—CH₂—C≡CR^(h)). The terms “alkylene,” “alkenylene” and “alkynylene”refer to bivalent forms of alkyl, alkenyl and alkynyl groups,respectively.

The groups set forth above, can be substituted with a wide variety ofsubstituents to synthesize homocamptothecin analogs retaining activity.For example, alkyl groups may preferably be substituted with a group orgroups including, but not limited to, a benzyl group, a phenyl group, analkoxy group, a hydroxy group, an amino group (including, for example,free amino groups, alkylamino, dialkylamino groups and arylaminogroups), an alkenyl group, an alkynyl group and an acyloxy group. In thecase of amino groups (—NR^(a)R^(b)), R^(a) and R^(b) are preferablyindependently hydrogen, an acyl group, an alkyl group, or an aryl group.Acyl groups may preferably be substituted with (that is, R^(f) is) analkyl group, a haloalkyl group (for example, a perfluoroalkyl group), analkoxy group, an amino group and a hydroxy group. Alkynyl groups andalkenyl groups may preferably be substituted with (that is, R^(g) andR^(h) are preferably) a group or groups including, but not limited to,an alkyl group, an alkoxyalkyl group, an amino alkyl group and a benzylgroup.

The term “acyloxy” as used herein refers to the group —OC(O)R^(d).

The term “alkoxycarbonyloxy” as used herein refers to the group—OC(O)OR^(d).

The term “carbamoyloxy” as used herein refers to the group—OC(O)NR^(a)R^(b).

Amino and hydroxy groups may include protective groups as known in theart. Preferred protective groups for amino groups includetert-butyloxycarbonyl, formyl, acetyl, benzyl,p-methoxybenzyloxycarbonyl, trityl. Other suitable protecting groups asknown to those skilled in the art are disclosed in Greene, T., Wuts, P.G. M., Protective Groups in Organic Synthesis, Wiley (1991), thedisclosure of which is incorporated herein by reference.

In general, R¹, R², R³, R⁶, R⁷ and R⁸ are preferably not excessivelybulky to maintain activity of the resultant camptothecin analog.Preferably, therefore, R¹, R², R³, R⁶, R⁷ and R⁸ independently have amolecular weight less than approximately 250. More preferably R¹, R²,R³, R⁶, R⁷ and R⁸ independently have a molecular weight less thanapproximately 200.

Some of the camptothecin analogs of the present invention can beprepared for pharmaceutical use as salts with inorganic acids such as,but not limited to, hydrochloride, hydrobromide, sulfate, phosphate, andnitrate. The camptothecin analogs can also be prepared as salts withorganic acids such as, but not limited to, acetate, tartrate, fumarate,succinate, citrate, methanesulfonate, p-toluenesulfonate, and stearate.Other acids can be used as intermediates in the preparation of thecompounds of the present invention and their pharmaceutically acceptablesalts.

For purification, administration or other purposes, the E-ring (thelactone ring) may be opened with alkali metal such as, but not limitedto, sodium hydroxide or calcium hydroxide, to form opened E-ring analogsof compounds of formula (1) as set forth in the compounds of formula(2). The intermediates thus obtained are more soluble in water and maybe purified to produce, after treatment with an acid, a purified form ofthe camptothecin analogs of the present invention.

The E-ring may also be modified to produce analogs of compounds offormula (1) with different solubility profiles in water or othersolvents. Methods to achieve this goal include, but are not limited to,opening the E-ring with hydroxide or a water-soluble amino group orfunctionalizing the hydroxy group at position 20 of the E-ring with awater-soluble group such as a polyethylene glycol group or an acylgroup. Such groups can be introduced either on the homocamptothecinderivative or at an earlier stage in the synthesis. The analogs thusprepared act as pro-drugs. In other words, these analogs regenerate thecompounds of formula (1) (with the closed E-ring structure) whenadministered to a living organism. See, Greenwald, R. B. et al., J. Med.Chem., 39, 1938 (1996). Alkyl esters resulting from acylation at C20,for example, will result in more lipophilic pro-drugs that may nothydrolyze until the alkyl group is enzymatically cleaved.

The present invention also provides a method of treating a patient,which comprises administering a pharmaceutically effective amount of acompound of formulas (1) and/or (2) or a pharmaceutically acceptablesalt thereof. The compound may, for example, be administered to apatient afflicted with cancer and/or leukemia. The compounds of thepresent invention may also act as antiviral (for example, anti-HIV)agents and antiparasitic agents. The compounds of formulas (1) and/or(2) may be administered by any conventional route of administration,including, but not limited to, intravenously, intramuscularly, orally,subcutaneously, intratumorally, intradermally, and parenterally. Thepharmaceutically effective amount or dosage is preferably between 0.01to 60 mg of one of the compounds of formulas (1) and (2) per kg of bodyweight. More preferably, the pharmaceutically effective amount or dosageis preferably between 0.1 to 40 mg of one of the compounds of formulas(1) and (2) per kg of body weight. In general, a pharmaceuticallyeffective amount or dosage contains an amount of one of the compounds offormulas (1) and/or (2) effective to display antileukemic, antitumor(anticancer), antiviral and/or antiparisitic behavior. Pharmaceuticalcompositions containing as an active ingredient one of the compounds offormulas (1) and/or (2) or a pharmaceutically acceptable salt thereof inassociation with a pharmaceutically acceptable carrier or diluent arealso within the scope of the present invention.

The present invention also provides a pharmaceutical compositioncomprising any of the compounds of formulas (1) and (2) and apharmaceutically acceptable carrier. The composition may, for example,contain between 0.1 mg and 3 g, and preferably between approximately 0.1mg and 500 mg of the compounds of formulas (1) and/or (2), and may beconstituted into any form suitable for the mode of administration.

The structural modifications of the present invention were found toprevent high affinity binding between the carboxylate form of acamptothecin analog and HSA, while at the same time promoting lactoneinteractions with erythrocytes. An additional consideration in thedesign of plasma and blood-stable camptothecins concerns the structureof the E-ring. The A,B,E- or B,E-ring modified camptothecins of thepresent invention: 1) display enhanced stability in the presence of HSAthrough elimination or minimization of the highly preferential bindingby HSA of carboxylate over lactone forms; 2) display high levels oflipophilicity which promote reversible associations of the lactone formsof the drugs with red blood cells, thereby slowing and restricting theextent of drug hydrolysis; and 3) display improved stability in aqueoussolution.

We further discovered that the novel blood-stable silyl-substitutedhomocamptothecin (referred to herein as β-hydroxylactone silatecans orhomosilatecans (hST)) derivatives of the present invention can beprepared by significant modification of a total synthesis approaches setforth in U.S. patent application Ser. No. 09/212,178, entitledCAMPTOTHECIN ANALOGS AND METHODS OF PREPARATION THEREOF and filed Dec.15, 1998 and U.S. patent application Ser. No. 09/007,872, entitled NOVELINTERMEDIATES IN THE SYNTHESIS OF CAMPTOTHECIN AND RELATED COMPOUNDS ANDSYNTHESIS THEREOF and filed Jan. 15, 1998 the disclosure of which areincorporated herein by reference. Novel intermediates were synthesizedto carry out the cascade radical annulation of the present invention.

Several model compounds of the present invention, as described in theformula below, were studied extensively.

R⁶ R² name 1b SiMe₂ ^(t)Bu NH₂ DB-90 1d SiMe₃ NH₂ DB-38 1f SiMe₂ ^(t)BuOH DB-91 1g SiMe₂ ^(t)Bu H DB-81 1h SiMe₃ H DB-33The novel homocamptothecins of the present invention contain A,B- orB-ring modifications which decrease the preferential carboxylate overlactone binding by human albumin. These modifications in the A,B-ringsalso markedly enhance lipophilicity and promote lactone associationswith lipid bilayers present in blood. The new compounds also contain anexpanded β-hydroxylactone E-ring which improved the overall stability ofthe agents without loss of potency. In cytotoxicity assays usingMDA-MB-435 breast cancer cells, the E-ring expanded β-hydroxylactonesilatecans of the present invention display IC₅₀ values in the range of2 to 115 nM. The compounds of the present invention (several of whichare described in the formula above), as a result of their novelstructural substitutions, have superior human plasma and human bloodstabilities than the agents described by Layergne et al.

Synthesis of the novel A,B,E-ring modified and B,E-ring modifiedcamptothecins of the present invention has led to the identification ofthe most blood-stable camptothecins displaying intrinsic potency yet tobe identified. An additional benefit of these new agents is that they donot display any significant interspecies variations in blood stabilitiessuch as those of 9-AC and camptothecin described in Mi, Z. and Burke, T.G. “Marked Interspecies Variations Concerning the Interactions ofCamptothecin with Serum Albumins: A Frequency-Domain FluorescenceSpectroscopic Study”, Biochemistry, 33: 12540-12545 (1994). This veryattractive feature should greatly facilitate the drug developmentprocess and the translation of experimental observations and dosingschedules developed in animal models to the clinic.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates synthesis of precursors for the cascade radicalreactions.

FIGS. 2 a and 2 b illustrate synthesis of new AB-ring modifiedhomocamptothecin/homosilatecan derivatives.

FIG. 3 illustrates a typical fluorescence fluorescence emission spectrafor a homosilatecan (1 μM 7-trimethylsilyl-10-aminohomocamptothecin(DB-38)) in the presence and absence of lipid bilayer membranes.

FIG. 4 illustrates a comparison of the equilibrium binding of four novelhomosilatecans to SUVs composed of electroneutraldimyristoylphosphatidylcholine (DMPC) in PBS with data acquired forcamptothecin (CPT) and topotecan (TPT) as well.

FIG. 5 illustrates the marked dependence of the fluorescence emissionspectra of 1 μl 7-t-butyldimethylsilyl-10-hydroxyhomocamptothecin(DB-91) on the presence of water.

FIG. 6 illustrates the fluorescence emission spectra of 1 μl7-t-butyldimethylsilyl-10-hydroxy-homocamptothecin (DB-91) in solutionsof phosphate-buffered saline (PBS) at pH 7.4 and in PBS at pH 7.4containing albumin-free red blood cells at a concentration of (10±1)×10⁶cell/μL.

FIG. 7 illustrates the fluorescence emission spectra of prior artcompound 7-ethyl-10-hydroxycamptothecin (SN-38) in solutions ofphosphate-buffered saline (PBS) at pH 7.4 and in PBS at pH 7.4containing albumin-free red blood cells at a concentration of (10±1)×10⁶cell/μL.

FIG. 8 illustrates the pH dependence of the stability of 1 μM7-trimethylsilyl-10-aminohomocamptothecin (DB38) in solutions ofphosphate-buffered saline (PBS) at pH values of 5.0, 7.4, 8.0, and 9.0as determined using HPLC methods.

FIG. 9 illustrates the pH dependence of the stability of 1 μM7-t-butyldimethylsilylhomocamptothecin (DB81) in solutions ofphosphate-buffered saline (PBS) at pH values of 5.0, 7.4, 8.0, and 9.0as determined using HPLC methods.

FIG. 10 illustrates the pH dependence of the stability of 1 μM7-t-butyldimethylsilyl-10-aminohomo-camptothecin (DB90) in solutions ofphosphate-buffered saline (PBS) at pH values of 5.0, 7.4, 8.0, and 9.0as determined using HPLC methods.

FIG. 11 illustrates the pH dependence of the stability of 1 μM of7-t-butyldimethylsilyl-10-hydroxyhomo-camptothecin (DB91) in solutionsof phosphate-buffered saline (PBS) at pH values of 5.0, 7.4, 8.0, and9.0 as determined using HPLC methods.

FIG. 12 illustrates the improved stabilities of four novelhomosilatecans of the current invention in PBS solution as determinedusing HPLC methods.

FIG. 13 depicts the improved stabilities of four novel homosilatecans ofthe current invention in PBS/HSA as determined by HPLC methods.

FIG. 14 depicts the human plasma stabilities of four novelhomosilatecans of the current invention as determined using HPLCmethods.

FIG. 15 depicts the stabilities of four novel homosilatecans of thecurrent invention in PBS suspensions containing physiologically-relevantconcentrations [(5±1)×10⁶ cell/μL] of albumin-free red blood cells.

FIG. 16 illustrates the improved human blood stabilities of four novelhomosilatecans of the current invention as determined using HPLCmethods.

FIG. 17 illustrates the improved human blood stabilities of four novelhomosilatecans of this invention relative to clinically-relevant agentsof the prior art which include 9-aminocamptothecin (9AC), camptothecin(CPT), topotecan (TPT) and SN-38.

DETAILED DESCRIPTION OF THE INVENTION Method of Preparation

The compounds of formula I in the present invention can be preparedaccording to the synthetic schemes outlined in FIGS. 1 and 2. FIG. 1shows the synthesis of a key iodopyridone 9, which can be used to makethe compounds of formula 1. The synthesis of 9 starts from enol ether 3,an intermediate in the synthesis of camptothecin and analogs. See U.S.patent application Ser. No. 09/007,872, the disclosure of which isincorporated herein by reference. Dihydroxylation followed by oxidativecleavage provides the keto formate 5, which is then extended by aReformatsky reaction to give 6. Conveniently, the formyl group iscleaved in this reaction, and acid promoted cleavage of the t-butylester directly results in β-hydroxylactone 7. This compound is thenconverted to the iodopyridone 9 by a sequence of 1) iodinativedesilylation, and 2) demethylation.

FIG. 2 a shows the conversion of iodopyridone 9 to several model ABmodified homocamptothecin derivatives. N-Propargylation of 9 underoptimized conditions provides radical precursors 10a,b. The cascaderadical annulations of these precursors with the indicated isonitrilesgive products 1a,c,e,g,h. Products 1a,c,e were then deprotected bystandard means to provide the target drug candidates 1b,d,f in theindicated overall yields. Compounds 1g and 1 h do not requiredeprotection. The synthesis of additional novel compounds of the presentinvention are described in the Examples section of the presentapplication.

Layergne and coworkers disclose two ways to make homosilatecanderivatives, but both have serious limitations. The first involves theconversion of a standard camptothecin derivative to a homocamptothecinderivative by a series of steps involving disassembly of the normallactone and reassembly of the homologated lactone. This route is limitedbecause it requires an existing camptothecin to start. Furthermore, manyexisting camptothecin derivatives bear substituents that would not beexpected to survive the harsh conditions of the refashioning of thelactone. This process requires reducing, oxidizing, strong acid, andstrong base steps. The second route involves a total synthesis using apalladium catalyzed cyclization to form ring C. This route is limited bythe availability of A-ring precursors and by the ability of thesubstituents thereon to survive the many subsequent steps of thesynthesis. Furthermore, the synthesis does not appear to offer thepossibility to introducing many B ring substituents, including thesubstituents described herein.

In contrast, the radical cascade synthetic schemes of the presentinvention are much more tolerant and flexible and can be used to makehomocamptothecin derivatives with many A-, B-, or A/B substitutionpatterns, as shown, for example, in FIG. 2 b. Generally, variousreagents can be used in the radical cascade including, but not limitedto, hexamethylditin, hexamethyldisilane, ortetrakis(trimethylsilyl)silane. The source of energy for this reactioncan be a sun lamp or an ultraviolet lamp. The temperature is preferablyset between approximately 25 and 150° C. More preferably, thetemperature is set at approximately 70° C. There are generally nolimitations upon the choice of solvent used other than inertness to theradical cascade. Preferred solvents include benzene, toluene,benzotrifluoride, acetonitrile, THF and tert-butanol. Also, there isvery broad latitude in the choice of substituents on the alkyne (R⁶) andthe isonitrile (R¹-R⁴) in the synthetic schemes of the present inventionbecause of the mildness of the reaction conditions. In addition, in rarecases where suitable propargyl derivatives are not readily prepared,allyl derivatives can be substituted instead and the same final productsare formed, albeit in lower yield.

The substituent on C20 (R⁵) can also be widely varied since it derivesfrom readily available allyl alcohols. Substituted esters can also beused in the Reformatsky reaction to provide compounds with substitutentson C20a (R¹³). While the compounds of this invention might be used inracemic form for chemotherapy, it is more preferable to use samples thatare exclusively or predominately the biologically active enantiomer atC20. Because of a change in priorities in the Cahn-Ingold-Prelogs Rulesfor assignment of absolute configuration, the C20 S enantiomer of astandard camptothecin generally has the same relative configuration asthe C20 R enantiomer of the corresponding homocamptothecin. Racemic orenantiomerically enriched samples of homocamptothecin derivatives can beseparated into their individual components by standard methods of liquidchromatography using commercially available chiral columns. SeeLayergne, O.; et al., “Homocamptothecins: Synthesis and AntitumorActivity of Novel E-Ring Modified Camptothecin Analogs,” J. Med. Chem.,41, 5410-5419 (1998).

Human Blood Stabilities of Camptothecins and the Basis for the RationalDesign of Blood-Stable Homosilatecans with High Potency.

Recently the intrinsic fluorescent emissions from the lactone andcarboxylate forms of camptothecin and related analogs have been studiedin order to elucidate their markedly different interactions with humanblood components. Burke, T. G. and Mi, Z., “Ethyl substitution at the 7position extends the half-life of 10-hydroxycamptothecin in the presenceof human serum albumin,” J. Med. Chem. 36: 2580-2582 (1993); Burke, T.G., Mishra, A. K., Wani, M. C. and Wall, M. E., “Lipid bilayerpartitioning and stability of camptothecin drugs,” Biochemistry. 32:5352-5364 (1993); Burke, T. G. and Mi, Z.: “Preferential Binding of theCarboxylate Form of Camptothecin by Human Serum Albumin,” (1993a) Anal.Biochem. 212, 285-287; Burke, T. G. and Mi, Z., “The Structural Basis ofCamptothecin Interactions with Human Serum Albumin: Impact on DrugStability,” (1994) J. Med. Chem. 37, 40-46; Burke, T. G. Munshi, C. B.,Mi, Z., and Jiang, Y., “The Important Role of Albumin in Determining theRelative Human Blood Stabilities of the Camptothecin Anticancer Drugs,”(1995) J. Pharma. Sci. 84, 518-519; Mi, Z. and Burke, T. G.,“Differential Interactions of Camptothecin Lactone and Carboxylate Formswith Human Blood Components,” (1994a) Biochemistry, 33, 10325-10336; Mi,Z. and Burke, T. G., “Marked Interspecies Variations Concerning theInteractions of Camptothecin with Serum Albumins: A Frequency-DomainFluorescence Spectroscopic Study,” (1994b) Biochemistry 33, 12540-12545;and Mi, Z., Malak, H., and Burke, T. G., “Reduced Albumin BindingPromotes the Stability and Activity of Topotecan in Human Blood,” (1995)Biochemistry, 34, 13722-13728, the disclosures of which are incorporatedherein by reference.

In phosphate buffered saline (PBS) at pH 7.4, frequency-domainfluorescence lifetime spectroscopy reveals that human serum albumin(HSA) preferentially binds the carboxylate form of camptothecin with a200-fold higher affinity than the lactone form. These interactionsresult in camptothecin opening more rapidly and completely in thepresence of HSA than in the absence of the protein. In human plasma, pH7.4 and 37° C., camptothecin lactone opens rapidly and completely to thecarboxylate form with a t_(1/2) value of 11 min and an almost negligiblepercentage of lactone at equilibrium value of 0.2%. In whole bloodversus plasma, camptothecin displayed enhanced stability (t_(1/2) valueof 22 min and a percentage of lactone at equilibrium value of 5.3%). Theenhanced stability of camptothecin lactone in human blood was found tobe a result of drug associations with the lipid bilayers of red bloodcells. Camptothecin binds erythrocyte membranes, the drug localizeswithin the acyl chain region, and accordingly remains protected fromhydrolysis.

The human blood stabilities of the several camptothecin analogs ofclinical interest have also been compared. As was observed in the caseof camptothecin, 9-aminocamptothecin (9-AC) was observed to hydrolyzealmost completely (>99%) in PBS solution containing HSA. Although noattempt was made to spectroscopically quantify the relative bindingaffinities of the lactone and carboxylate forms of the 9-amino congener(because of the significantly reduced fluorescence quantum yields of9-AC lactone and carboxylate species relative to camptothecin), HPLCdata were consistent with HSA preferentially binding the carboxylateform of this agent over its lactone form. In plasma it was observedthat >99.5% of the 9-amino analog of camptothecin converted tocarboxylate, a finding which again closely parallels stability dataobtained using camptothecin. In whole blood, <0.5% and 5.3% are thefractions of 9-aminocamptothecin and camptothecin, respectively, whichremained in the lactone form at equilibrium. The approximately 10-foldhigher level of lactone remaining at equilibrium for camptothecinrelative to 9-aminocamptothecin may, in part, be accounted for by theenhanced lipophilicity and greater ability of camptothecin to transitionfrom the aqueous environment and into erythrocyte membranes present inwhole blood.

In contrast to the low levels of lactone remaining at equilibrium inwhole human blood for camptothecin and 9-aminocamptothecin (<0.5% and5.3%, respectively), topotecan (11.9%), CPT-11 (21.0%), and SN-38(19.5%) all display improved blood stabilities. While lactone levels atequilibrium for topotecan are 20-fold greater than for9-aminocamptothecin, the corresponding levels of lactone for CPT-11 andSN-38 are approximately 40-fold greater than in the case of9-aminocamptothecin. The significant gains in the relative stabilitiesof topotecan, CPT-11, and SN-38 can be correlated to their favorableinteractions with HSA. It is believed that structural substituents atthe 7- and 9-positions hinder and prevent the preferential binding ofthe carboxylate drug forms by HSA. The technique of time-resolvedfluorescence anisotropy has recently been used to demonstrate that,under experimental conditions where camptothecin carboxylate associateswith HSA and tumbles in solution closely associated with the protein,the carboxylate forms of topotecan and CPT-11 do not associate with HSA.In the case of SN-38, direct spectroscopic evidence has been obtainedwhich indicates that HSA preferentially binds the lactone form of thisagent, thereby shifting the lactone-carboxylate equilibrium to thelactone.

These observations indicate that HSA plays an important role indetermining the relative human blood stabilities of the camptothecins.In the cases of camptothecin and 9-aminocamptothecin, the protein actsas a sink for the carboxylate drug form, binding the opened ring speciesand thereby shifting the lactone-carboxylate equilibria to the right.However, in the cases of topotecan, CPT-11, and SN-38, no suchpreferential binding of the carboxylate drug form by HSA is observed.Opposite to the situation with camptothecin and its 9-amino analogue,HSA preferentially binds the lactone form of SN-38 which therebypromotes higher circulatory levels of this biologically active species.

The rapid and extensive loss of active drug that occurs with clinicallyrelevant camptothecins indicates that it would be highly advantageous toidentify camptothecins with improved human blood stabilities.

In the present studies we modified camptothecin in the A and B ringswith the effect of: 1) reducing protein binding; 2) enhancinglipophilicity; and 3) producing both a concomitant reduction incarboxylate binding to human albumin while also enhancing lipophilicity.We also included an expanded E-ring in the design of the presentcompounds. Our studies have led to the design of novel A,B,E-ringmodified camptothecins which are the most blood-stable and intrinsicallypotent camptothecin analogs yet to be identified, with blood stabilityparameters outcompeting the prior art compound homocamptothecin as wellA,B-ring modified camptothecin analogs containing a conventionalα-hydroxylactone functionality. The novel camptothecin analogs of thepresent invention display unique properties such as superior human bloodstabilities in combination with high anticancer activities.

Fluorescence Anisotropy Titration Demonstrates that the NovelHomosilatecans of the Present Invention Display a Broad Range ofEquilibrium Association Constants for Lipid Vesicles and that E-RingExpansion Enhances the Lipophilicity of Silatecans.

FIG. 3 depicts the fluorescence emission spectra of 1 μM DB-38 inphosphate buffered saline (PBS) and in lipid bilayers. The data indicatethat upon introduction of lipid bilayers into the sample there is anincrease in the fluorescence emission of the compound, indicative of aninteraction between the drug and the membrane. Upon changing the solventto ethanol the fluorescence also changes. In each case with membranesthere is a marked increase in fluorescence intensity as the drugpartitions into the lipid bilayer microenvironment. In each case thereis also a prominent blue-shifting or shift in the emission spectra tolower wavelength upon drug interaction with membrane (see Table 1). Thespectral data presented in FIG. 3 indicate that homosilatecans arefluorescent and that the spectral parameters of the drugs change uponaddition of lipid bilayer membranes to the samples. Table 1 found belowcompares the maximum excitation and emission wavelengths of the newhomosilatecan analogs. We also examined the membrane interactions of thering-opened forms of DB-90 and DB-91, and our results indicate thesimilar spectral shifting for the ring-opened species.

TABLE 1 Fluorescence Spectral Parameters for Homosilatecans (DB-38,DB-81, DB-90, DB-91) in Solution and Bound to DMPC and DMPG SUVs.Excitation Compound (nm) Emission (nm) (S) PBS PBS DMPC DMPG DB-38 410531 515 517 DB-81 380 452 443 442 DB-90 402 535 513 512 DB-91 394 554441 426

The intrinsic fluorescent nature of the lactone and carboxylate forms ofhomosilatecans allows for the sensitive method of steady-statefluorescence anisotropy titration to be employed to determine thestrength of the binding interactions of the various analogs with lipidbilayers.

A steady-state fluorescence anisotropy (a) measurement is related to therotational rate of the fluorescent molecule through the Perrin Equation:

a _(o) /a=1+(τ/φ)

where a_(o) is the limiting fluorescence anisotropy in the absence ofdepolarizing rotations, τ is the excited-state lifetime, and φ is therotational correlation time of the fluorophore. The above equationstates that changes in either the τ or φ values of a fluorescentcompound can modulate its steady-state anisotropy.

The excited-state lifetime values of camptothecin in PBS, glycerol, andmethanol were examined at 37° C. The lifetime values were determined tobe 4.7 ns, 3.8 ns, and 3.5 ns, respectively. Similarly, the lifetimevalue of camptothecin when associated with DMPC bilayers was measured at37° C., and the average value for membrane-bound drug was found to be3.7 ns.

Thus the lifetime measurements described above indicate that theexcited-state lifetime of camptothecin is relatively insensitive toalterations in microenvironment (for example, a change in solvent orfluorophore relocation from an aqueous milieu to a phospholipidmembrane). For a fluorophore having a τ value that remains relativelyconstant during a transition which strongly impacts on its rotationalmotion (such as a change in solvent viscosity or fluorophore binding tolarge macromolecular assemblies such as liposomal particles), the Perrinequation indicates a direct relationship between a and φ values willexist (that is, as the φ value of the fluorescent compound increases,then so too does its steady-state anisotropy value).

It has been shown that the steady-state fluorescence anisotropy valuesof camptothecin analogs and novel homosilatecans are highly sensitive tosolvent viscosity and to associations with small unilamellar lipidvesicles. For example, topotecan has an a value of 0.008 in PBS, but itsa value increases 9-fold and 40-fold in the viscous solvents octanol andglycerol, respectively. A 21-fold enhancement in the a value ofcamptothecin is observed upon binding of drug to vesicles composed ofeither DMPC or DMPG. Because of the sensitivity of a of the camptothecindrugs to membrane associations, the method of fluorescence anisotropytitration was employed to study the equilibrium binding of camptothecinanalogs with lipid bilayers. The experiment includes determining the avalues for a set of samples where the drug concentration in each washeld constant (typically 1 or 2 μM), while the lipid concentration amongthe members of a set was varied from 0 to 0.29 M.

As a consequence of the brilliant fluorescence emissions from the newlysynthesized homosilatecans (a summary of the spectral parameters can befound in Table 1), the lipid bilayer adsorption isotherms summarized inFIG. 4 were relatively free from any background signal. The method offluorescence anisotropy titration was used to construct the adsorptionisotherms. The experiments were conducted at drug concentrations of 1 μMin PBS buffer (37° C.). The anisotropy values of DB-38, DB-90 and DB-91titrated much more rapidly than those of camptothecin or topotecan,indicating that the novel homosilatecans have much stronger interactionswith these membranes than camptothecin and topotecan. Because of thepotential of the lactone ring of the homosilatecans and camptothecins tohydrolyze in PBS, anisotropy values at each lipid concentration weredetermined immediately (approx. 1 min.) following the addition of thelactone form of each agent to the liposome suspension as to minimize anypossibility of conversion to the carboxylate form. Using drugconcentrations of 1 μM and long pass filters to isolate emitted lightfrom background signal (that is, scattered exciting light and extraneousfluorescence signal resulting from the possible presence of impurities),signal levels from drugs dissolved in PBS buffer were typically 99.97%in the absence of membrane and greater than 98% in the presence ofmembrane. Adsorption isotherms were used to determine overallassociation constants for the homosilatecan, silatecan, and camptothecindrugs. Overall association constants are defined as:

K=[A _(B) ]/[A _(F) ][L]

where [A_(B)] represents the concentration of bound drug, [A_(F)]represents the concentration of free drug, and [L] represents the totallipid concentration in the vesicle suspension. This equation is validwhen the concentration of free lipid is approximately equal to theconcentration of total lipid (that is, the concentration of free lipidis in significant excess over the concentration of bound drug). Providedthis condition is satisfied, K may be determined from the inverse of theslope of a double reciprocal plot. In such a double reciprocal plot,1/fraction of the total drug bound is plotted vs. 1/lipid concentration,with a y-intercept value of 1 (for a system displaying binding sitehomogeneity). Such double-reciprocal plots for the associations of thenew homosilatecans analogs (both lactone and carboxylate forms) withDMPC and DMPG small unilamellar vesicle (SUV) preparations were linearwith good correlation coefficients. The linearity of these plots, aswell as the corresponding plots for drug associations with other typesof membrane preparations, indicates that fluorophore binding at theselipid concentrations is adequately described by the above equation.

The studies summarized in Table 2 examine the structural basis ofhomosilatecan associations for lipid bilayers. Two types of membraneswere included in these studies which were conducted under nearphysiological conditions of pH and temperature; these membranes includefluid-phase and electroneutral L-α-dimyristoylphosphatidyl-choline(DMPC); and fluid-phase and negatively-chargedL-α-dimyristoylphosphatidylglycerol (DMPG). DMPC and DMPG have identicalchain length but the charges on their head groups differ.

TABLE 2 Overall association constants for homosilatecans andcamptothecin analogs interacting with unilamellar vesicles ofelectroneutral DMPC and negatively charged DMPG in PBS at pH 7.4 and 37°C. Compound K_(DMPC) (M⁻¹) K_(DMPG) (M⁻¹) DB-38 1400 800 DB-81 1440018500 DB-90 8600 9300 DB-91 8000 4300 DB-90 carboxylate form 770 80DB-91 carboxylate form 700 100 Topotecan 10 50 Camptothecin 100 100

In the studies of Table 2, binding isotherms were constructed using themethod of fluorescence anisotropy titration as discussed above, and Kvalues were determined from the slopes of the double-reciprocal plots.The K values are subject to 10% uncertainty. One of the most strikingfeatures of the data contained in Table 2 is the strong modulation whichcan be achieved through the creation of A,B,E-ring modifiedcamptothecins (for example, the homosilatecans known as DB-38, DB-90,and DB-91) or B,E-ring modified camptothecins (for example, thehomosilatecan known as DB-81). Homosilatecans containing either a solesubstitution at the 7 position or dual substitution at the 7 and 10positions have been created and found to display very highlipophilicities. Included in Table 2 are camptothecin compounds(topotecan and camptothecin). For DB-81, the lipophilicity for DMPCmembranes relative to corresponding value for topotecan increases over1,400-fold. Data for these agents were included to show the highlylipophilic nature of the new homocamptothecins relative to compoundssuch as topotecan and camptothecin. From Table 2, it is clear that thecompounds of the present invention are much more lipophilic than eithercamptothecin or topotecan.

Other interesting and unexpected findings are apparent upon inspectionof the data contained in Table 2. Comparison of the K_(DMPC) values fortwo homosilatecan with their corresponding silatecan analogs (where theE-ring systems are β-hydroxylactone versus α-hydroxylactone moieties,respectively) indicate that the homocamptothecins display greaterlipophilicity. For example, the K_(DMPC) values of the silatecancounterparts of DB-38 (CHJ-792, K_(DMPC)=820 M⁻¹) and DB-91 (DB-67,K_(DMPC)=2,500 M⁻¹) are approximately 2-fold to 3-fold less than for thecorresponding homosilatecans. Thus, for DB-38 and DB-91 the expandedE-ring is a favorable consideration for membrane binding which, in turn,promotes drug stability in human blood.

Another surprising trend was observed for the homosilatecans when thecarboxylate forms of the drugs were studied. A 3-fold decrease in theaffinity for DMPC upon the opening of the lactone ring of camptothecinhas previously been observed. Burke, T. G., Mishra, A. K., Wani, M. C.and Wall, M. E., “Lipid bilayer partitioning and stability ofcamptothecin drugs,” Biochemistry. 32: 5352-5364 (1993). For the DB-90and DB-91 homocamptothecins, we observe a 10-fold decrease in DMPCbinding upon ring opening. Hence, the homosilatecans not only displaymarkedly enhanced lipophilicity but the levels of differential bindingbetween the lactone and carboxylate forms appear to be significantlygreater (10-fold versus 3-fold) relative to camptothecins containingα-hydroxylactone ring systems. The two considerations described above(high lipophilicity and higher differential binding of lactone overcarboxylate forms) are contributing factors to the optimized bloodstabilities which the homosilatecans display over camptothecin andhomocamptothecin.

Direct Fluorescence Spectral Assessment of the Extensive MembraneInterations of the DB-91 Homosilatecan with Red Blood Cells.

FIG. 5 illustrates the fluorescence emission spectra of 1 μM7-t-butyldimethylsilyl-10-hydroxy-homocamptothecin (DB-91) in solutionsof phosphate-buffered saline (PBS) at pH 7.4, ethanol, and admixturesthereof. All spectra were recorded using exciting light of 394 nm at 37°C. The emission maxima for DB-91 in PBS is 554 nm, but this value shiftssignificantly to a λ_(max) of approximately 410 nm in anhydrous ethanol.Because DB-91 contains a 10-hydroxy functionality, the possibilityexists that fluorescence can occur from two distinct species. In anaprotic solvent or non-aqueous microenvironment a protonated (withrespect to the 10-hydroxy functionality) species predominates, while inprotic solvents such as water a deprotonated excited-state complexpredominates. The 554 nm peak is correlated with the deprotonatedexcited-state complex while the λ_(max) of approximately 410 nmcorrelates with the protonated excited-state complex. The formation ofthe deprotonated excited-state complex is greatly facilitated by thepresence of water; even at small amounts of water such as 1% a peak isapparent around 550 nm which correlates with the water-facilitatedformation of the deprotonated excited-state complex. The spectralsensitivity of DB-91, and other members of the camptothecin familycontaining the 10-hydroxy functionality, provides a useful approach forstudying the partitioning of drug from an aqueous environment into ahydrophobic environment such at the surface of a red blood cell.

FIG. 6 shows the fluorescence emission spectra of 1 μM7-t-butyldimethylsilyl-10-hydroxy-homocamptothecin (DB91) in solutionsof phosphate-buffered saline (PBS) at pH 7.4 and in PBS at pH 7.4containing albumin-free red blood cells at a concentration of (10±1)×10⁶cell/μL and provides direct evidence of the extensive interactions of ahomosilatecan with red blood cells. Spectra were recorded in front-facecuvettes (to optimize fluorescence versus scatter levels) at 37° C.using a DB-91 concentration of 10 μM and exciting light of 370 nm.

The emission maxima for DB-91 in PBS is 554 nm. In the presence of redblood cells, a peak with a significantly lower λ_(max) value is observedindicating that the agent is capable of partitioning into the red bloodcell membranes. The membranes of the red blood cells provide ahydrophobic microenvironment where the protonated excited-statecomplexes can form and fluoresce from. Comparison of the emissionspectra of DB-91 in the presence of human erythrocytes with that ofclinically relevant 7-ethyl-10-hydroxycamptothecin (SN-38), asillustrated in FIG. 7, indicate there is more extensive protonatedexcited-state complex formation in the case of DB-91. Similar to thestudies of FIG. 6, the spectra of FIG. 7 were recorded in front-facecuvettes at 37° C. using an SN-38 concentration of 10 μM and excitinglight of 370 nm These findings corroborate model membrane studiesindicating the membrane binding of SN-38 is significantly less than theextensive interactions noted for DB-91 (SN-38 displays a K_(DMPC) valueof 300 M⁻¹ whereas DB-91 displays a K_(DMPC) value of 8,000 M⁻¹). Fromour spectral studies we conclude the novel homosilatcan DB-91 is a morelipophilic, erythrocyte-interactive agent than the FDA-approved SN-38.

Homosilatecans Display Improved Stabilities in Aqueous Solution Relativeto Camptothecins Containing α-Hydroxylactone Pharmacophores.

FIGS. 8 through 11 illustrate the pH dependence of the stability of 1 μMsolutions of DB-38, DB-81, DB-90, and DB-91 in solutions ofphosphate-buffered saline (PBS) at pH values of 5.0, 7.4, 8.0, and 9.0.The stability parameters for each drug were determined using HPLCmethods. All experiments were conducted at 37° C. Hydrolysis is observedat pH values of 7.4, 8.0, and 9.0 with more extensive hydrolysis beingnoted at the higher pH values.

Although hydrolysis is observed at pH values of 7.4, 8.0, and 9.0, ourdata indicate that the lactone ring of homosilatecans is less labile(that is, significantly slower in undergoing hydrolysis) relative toboth silatecans and camptothecins containing the conventionalα-hydroxylactone ring moiety.

FIG. 12 contrasts the improved stabilities of four novel homosilatecansof the present invention with their corresponding silatecan structurecontaining the α-hydroxylactone functionality. All the experiments ofFIG. 12 were conducted in PBS at 37° C. Panels A through D each containstability profiles for a novel homosilatecan (open circles) and itscorresponding silatecan (solid circles) containing the conventionalα-hydroxylactone ring moiety found in camptothecin and other clinicallyrelevant camptothecin analogs such as topotecan, SN-38, CPT-11 and9-aminocamptothecin.

In all cases, the agents containing the expanded E-ring or homosilatecanstructures displayed markedly enhanced stability. The stabilityparameters for the homosilatecans are summarized in Table 3. The dataindicate that the lactone ring of homosilatecans is less labile (thatis, significantly slower in undergoing hydrolysis) relative to theα-hydroxylactone ring moiety contained in both silatecans andcamptothecins. For silatecans and camptothecins such as topotecan andcamptothecin, approximately 12% of lactone remains at equilibrium after3 hours, whereas greater than 80% lactone remains for each of thehomosilatecans under identical incubation conditions.

Determination of the Superior Stabilities of Homosilatecans in thePresence of Human Serum Albumin.

FIG. 13 depicts the improved stabilities of four novel homosilatecans ofthe current invention following incubation in PBS containing 30 mg/mlhuman serum albumin at 37° C. Panels A through D each contain stabilityprofiles for a novel homosilatecan and its corresponding silatecancontaining the conventional α-hydroxylactone ring moiety found incamptothecin and other clinically relevant camptothecin analogs such astopotecan, SN-38, CPT-11 and 9-aminocamptothecin. In all cases, theagents containing the expanded E-ring structures displayed markedlyenhanced stabilities in the presence of HSA. The stability parametersfor the homosilatecans are summarized in Table 3. As illustrated in FIG.14, the homosilatecans of the current invention also displayed superiorstabilities in human plasma than camptothecins such as topotecan, SN-38,and CPT-11. Of the homosilatecans, DB-81 displayed the highest stabilityin human plasma, followed by DB-90 and DB-91, with DB-38 (the leastlipophilic of the homosilatecans studied) displaying the loweststability in human plasma. All the experiments of FIG. 14 were conductedin human plasma at 37° C. Plasma samples were continuously aerated by astream of blood gas resulting in the maintenance of pH at values of7.5±0.1. In all cases, the agents containing the expanded E-ring orhomosilatecan structures displayed markedly enhanced stabilitiesrelative to the parent drug camptothecin containing the conventionalα-hydroxylactone ring moiety. The stability parameters for thehomosilatecans are summarized in Table 3.

Our studies demonstrate that both lipophilic as well as morewater-soluble homosilatecans display improved stabilities overhomocamptothecin that contains no substitutions in the A and B rings.See Layergne et al. “Homocamptothecins: Synthesis and Antitumor Activityof Novel E-Ring-Modified Camptothecin Analogues” J. Med. Chem. 41:5410-5419 (1998). Thus, the present invention indicates thatsubstitution of the A and B ring of homocamptothecin is a favorablefactor with respect to blood stabilities. The likely explanation is thatthe unsubstituted homocamptothecin carboxylate, like camptothecincarboxylate, binds HSA preferentially in the carboxylate form andeffectively shifts the lactone-carboxylate equilibria to the right.

Our results indicate that dramatically improved human plasma stabilitycan be realized by combining the β-hydroxylactone pharmacophore with thefollowing concomitant structural changes: 1) B-ring modification such asa silyl or an silylalkyl functionality at position 7 (e.g. DB-81); 2)A-ring modification such as the structural modifications contained intopotecan (water-solublizing changes such as inclusion of9-dimethylaminomethyl and 10-hydroxy functionalities disfavorcarboxylate binding to HSA; and 3) combined substitution in both the Aand the B ring that includes, for example, a silyl or silylalkylsubstituent at position 7 (e.g. DB-90 and DB-91). See also Mi, Z.,Malak, H., and Burke, T. G., “Reduced Albumin Binding Promotes theStability and Activity of Topotecan in Human Blood,” Biochemistry, 34,13722-13728 (1995). The compounds of the latter example display highlipophilicities and reduced specific interactions between thecarboxylate drug form and HSA, both factors contributing to improvedplasma stability.

Markedly Enhanced Stabilities of the Novel Homosilatecans in HumanBlood.

FIG. 15 depicts the stabilities of four novel homosilatecans of thecurrent invention in PBS suspensions containing physiologically-relevantconcentrations [(5±1)×10⁶ cell/μL] of albumin-free red blood cells.Stability characteristics were determined at 37° C. using HPLC methods.In all cases, the agents containing the expanded E-ring or homosilatecanstructures displayed markedly enhanced stabilities in the presence ofred blood cells relative to published literature values for camptothecinanalogs containing the conventional α-hydroxylactone ring moiety (suchas the clinically relevant agents SN-38, 9-aminocamptothecin,9-nitrocamptothecin, GI-147211C, topotecan, etc. The stabilityparameters for the homosilatecans are summarized in Table 3.

FIG. 16 and FIG. 17 depict the improved human blood stabilities of fournovel homosilatecans of the present invention. All experiments wereconducted at pH 7.4 and 37° C. In FIG. 16, panels A through D eachcontain stability profiles for a novel homosilatecan (open circles) andits corresponding silatecan (solid circles) containing the conventionalα-hydroxylactone ring moiety found in camptothecin and other clinicallyrelevant and camptothecin analogs such as topotecan, SN-38, CPT-11 and9-aminocamptothecin and experimental agents as well. In all cases, theagents containing the expanded E-ring or homosilatecan structuresdisplayed markedly enhanced human blood stabilities relative tocamptothecin analogs such as topotecan and SN-38. FIG. 17 illustratesthe improved human blood stabilities of the novel homosilatecans of thepresent invention compared to current clinically-relevant agentsincluding 9-aminocamptothecin (9AC), camptothecin (CPT), topotecan (TPT)and SN-38 (SN38). The stability parameters of FIGS. 16 and 17 aresummarized in Table 3.

The human blood stability values noted for DB-81, DB-90 and DB-91 arethe highest yet to be measured for a intrinsically potent camptothecinanalog. The greater than 80% lactone values following 3 hrs. ofincubation compare very favorably relative to the corresponding percentlactone levels in whole human blood for 9-aminocamptothecin (approx.0.3%), camptothecin (approx. 6%) topotecan (approx. 15%), CPT-11(approx. 21.0%), and SN-38 (approx. 30%).

TABLE 3 Summary of Human Blood Stability Parameters for HomosilatecansIncubatio DRUG NAME n Time % and FLUID (Hours) Lactone DB-38 Whole Blood3 56.4 ± 0.6 HSA 3 81.4 ± 0.3 24 34.1 ± 2.2 PBS 3 82.8 ± 0.7 24 26.8 ±2.3 Plasma 3 40.3 ± 2.1 RBC 3 84.8 ± 1.5 24 39.4 ± 1.2 DB-81 Whole Blood3 86.6 ± 0.5 24 27.0 ± 2.3 HSA 3 88.1 ± 0.2 24 42.5 ± 1.6 PBS 3 84.9 ±0.3 24 30.9 ± 2.0 Plasma 3 85.0 ± 4.3 RBC 3 92.0 ± 0.0 24 55.8 ± 1.7DB-90 Whole Blood 3 85.2 ± 0.7 24 24.6 ± 1.3 HSA 3 86.8 ± 0.2 24 42.0 ±2.7 PBS 3 83.7 ± 0.5 24 26.1 ± 1.0 Plasma 3 71.1 ± 3.5 RBC 3 85.5 ± 0.424 38.5 ± 1.4 DB-91 Whole Blood 3 84.9 ± 0.3 24 37.1 ± 1.8 HSA 3 82.9 ±0.3 24 33.2 ± 3.0 PBS 3 83.1 ± 0.3 24 32.2 ± 1.0 Plasma 3 61.5 ± 3.9 RBC3 88.5 ± 0.2 24 42.6 ± 2.6The Novel Homosilatecans of the Current Invention Overcome the MarkedInterspecies Variations with Respect to Blood Stabilities that have beenObserved in the Past for Clinically Relevant Camptothecins Such as9-Aminocamptothecin, 9-Nitrocamptothecin and Camptothecin.

Camptothecin and 9-aminocamptothecin, anticancer agents renown for theirnovel mechanism of action and outstanding murine in vivo activity, haveto date displayed only modest therapeutic utility against human cancers.The drugs contain the lactone ring moiety which, at pH 7.4, hydrolyzesto yield biologically-inactive carboxylate forms. Comparison of drugstabilities for 9-aminocamptothecin reveals that ring opening occurredto a much greater extent in human blood than mouse blood (see Table 4).Camptothecin has been shown previously to behave in a similar manner.Burke, T. G. Munshi, C. B., Mi, Z., and Jiang, Y., “The Important Roleof Albumin in Determining the Relative Human Blood Stabilities of theCamptothecin Anticancer Drugs,” J. Pharma. Sci. 84, 518-519 (1995); andMi, Z. and Burke, T. G., “Marked Interspecies Variations Concerning theInteractions of Camptothecin with Serum Albumins: A Frequency-DomainFluorescence Spectroscopic Study,” Biochemistry 33, 12540-12545 (1994).We have used the technique of multifrequency phase-modulationspectroscopic analyses of the intrinsic fluorescence emissions ofcamptothecin lactone and carboxylate to provide a physical explanationfor the extensive ring opening observed for camptothecin and9-aminocamptothecin in the presence of human serum albumin (HSA). HSAexhibits a marked 200-fold binding preference for the carboxylate(K=1.2×10⁶ M⁻¹) relative to the lactone (K≈5.5×10³ M⁻¹). Serum albuminsfrom other species were found to bind camptothecin carboxylate notnearly as tightly as HSA. Due to the unique capacity of human albumin tobind camptothecin carboxylate and 9-aminocamptothecin carboxylateresulting in extensive conversion of the drug to its biologicallyinactive form, it appears that the success of these agents ineradicating cancer in animal models may be inherently more difficult toduplicate in humans.

The data for the novel homosilatecans of the current invention showessentially only minor variations between lactone levels in mouse bloodversus human blood. The noted changes in mouse versus animal blood arevery small relative to the 100-fold difference in lactone levelsobserved for 9-aminocamptothecin. In mouse blood experiments for DB-81and DB-91, the lactone levels actually observed in human blood aremodestly underestimated by values of 6% and 20%, respectively. However,for 9-aminocamptothecin mouse blood overestimates by 100-fold thelactone levels actually observed in human blood. These results indicatethat there are fundamental physiological reasons to think that thesuccess of our novel homosilatecans in animal models can be more readilytranslated into humans relative to agents such as camptothecin and9-aminocamptothecin.

TABLE 4 Comparison of the Marked Interspecies Variations in BloodStabilities For Camptothecin and 9-Aminocamptothecin Versus theRelatively Minor Differences Observed for Novel, Highly LipophilicCamptothecin Analogs. ^(a) Percent Percent Ratio of Lactone in Lactonein Lactone Mouse Blood Human Blood Level after 3 Hours after 3 HoursMouse/ Compound of Incubation of Incubation Human 9-Aminocamptothecin 380.4 100 Camptothecin 20 7 3 DB-38 72 56 1.3 DB-81 80 87 0.9 DB-90 61 850.7 DB-91 70 85 0.8 ^(a) Experiments were conducted at pH 7.4 and 37° C.and lactone levels determined using HPLC methods. Blood samples weredrawn and kept at 5° C. prior to the initiation of an experiment.

Highly Lipophilic Camptothecins Display High Anticancer Potency Even inthe Presence Human Serum Albumin.

The cytotoxicities of various camptothecins against MDA-MB-435tumorigenic metastatic human breast cancer cells are summarized in Table5. The cytotoxicity values are for 72 hr. exposure times. Overall, wefound DB-38 to be the most potent of the four novel homosilatecans whichwe studied, with an IC₅₀ value of 20 nM, while the IC₅₀ values for theother homosilatecans ranged from 20 nM to 115 nM. Our results clearlyindicate that through novel homosilatecan development the stability ofthe agents in human and animal blood can be markedly improved andequalized without compromising the high intrinsic potency andcytotoxicity of this important class of anticancer drugs.

TABLE 5 IC₅₀ Values of Homosilatecans and Camptothecin Analogs AgainstMDA-MB-435 Tumorogenic Metastatic Human Breast Cancer Cells in theAbsence and Presence of Human Serum Albumin. IC₅₀ (nM) Compound (w/oHSA) Camptothecin 12 ± 4  DB-38 20 ± 3  DB-81 77 ± 13 DB-90 73 ± 8 DB-91 115 ± 5 

EXAMPLES Experimental Methods for the Qualitative and QuantitativeDetermination of Lipid Bilayer Partitioning (i.e. Lipophilicity) andLactone Ring Stability of the Novel Homosilatecans of the CurrentInvention

Chemicals. Camptothecin and topotecan were in their 20 (S)-configurationand were of high purity (>98%) as determined by HPLC assays withfluorescence detection. The preparation of the homosilatecans isdescribed elsewhere in this application. All other agents were reagentgrade and were used without further purification. High purity water wasprovided by a Milli-Q UV PLUS purification system (Bedford, Mass.) wasutilized in all experiments.

Drug Stock Solution Preparation. Stock solutions of the drugs wereprepared in dimethylsulfoxide (A.C.S. spectrophotometric grade, Aldrich,Milwaukee, Wis.) at a concentration of 2×10⁻³ M and stored in the darkat 4° C. L-α-Dimyristoylphosphatidylcholine (DMPC) andL-α-dimyristoylphosphatidylglycerol (DMPG) were obtained from AvantiPolar Lipids, Alabaster, Ala., and were used without furtherpurification. All other chemicals were reagent grade and were usedwithout further purification.

Vesicle Preparation. Small unilamellar vesicle (SUV) suspensions wereprepared the day of an experiment by a methodology reported previouslyBurke and Tritton, Biochemistry 24 5972-5980 (1985); and Burke, T. G.,Mishra, A. K., Wani, M. C. and Wall, M. E. “Lipid bilayer partitioningand stability of camptothecin drugs,” Biochemistry. 32: 5352-5364(1993), the disclosures of which are incorporated herein by reference.Briefly, stock lipid suspensions containing known amount of lipid (200mg/mL lipid or less) in phosphate buffered saline (PBS, pH 7.4) wereprepared by Vortex mixing for 5-10 min above the T_(M) of the lipid. Thelipid dispersions were then sonicated using a bath-type sonicator(Laboratory Supplies Co., Hicksville, N.Y.) for 3-4 h until they becameoptically clear. A decrease in pH from 7.4 to 6.8 was observed for theSUV preparations of DMPG; therefore, the pH of these SUV suspensions wasadjusted to 7.4 using small quantities of 2.5 M NaOH in PBS, followed byadditional sonication. Each type of vesicle suspension was annealed for30 min at 37° C. and then used in an experiment.

Fluorescence Instrumentation. Steady-state fluorescence measurementswere obtained on a SLM Model 9850 spectrofluorometer with a thermostatedcuvette compartment. This instrument was interfaced with an IBM PS/2model 55 SX computer. Excitation and emission spectra were recorded withan excitation resolution of 8 nm and an emission resolution of 4 nm. Inall cases spectra were corrected for background fluorescence and scatterfrom unlabeled lipids or from solvents by subtraction of the spectrum ofa blank. Steady-state fluorescence intensity measurements were made inthe absence of polarizers. Steady-state anisotropy (a) measurements weredetermined with the instrument in the “T-format” for simultaneousmeasurement of two polarized intensities. The alignment of polarizerswas checked routinely using a dilute suspension of 0.25 μm polystyrenemicrospheres (Polysciences, Inc, Warrington, Pa.) in water andanisotropy values of >0.99 were obtained. Alternatively, polarizerorientation was checked using a dilute solution of glycogen in water.The anisotropy was calculated from a=(I_(VV)−GI_(VH))/(L_(VV)+GI_(VH)),where G=I_(VH)/I_(HH) and the subscripts refer to vertical andhorizontal orientations of the excitation and emission polarizers,respectively.

Anisotropy measurements for homosilatecans and camptothecins wereconducted using exciting light of 370 to 400 nm and long pass filters oneach emission channel to isolate the drug's fluorescence signal frombackground scatter and/or residual fluorescence. All emission filterswere obtained from Oriel Corp (Stamford, Conn.). The combination ofexciting light and emission filters allowed adequate separation offluorescence from background signal. The contribution of backgroundfluorescence, together with scattered light, was typically less than 1%of the total intensity. Since the lactone rings of camptothecin andrelated congeners undergo hydrolysis in aqueous medium with half-livesof approximately 20 min., all measurements were completed within theshortest possible time (ca. 0.5 to 1 min) after mixing the drug stocksolution with thermally pre-equilibrated solutions such that theexperiments were free of hydrolysis product. In fluorescencespectroscopic experiments designed to provide information concerning theinteractions of homosilatecans and camptothecins with red blood cells,drug concentrations of 10 μM were used. Experiments with red blood cellswere carried out in front-face quartz cuvettes to optimize fluorescencesignal and minimize scattered light.

Determination of Equilibrium Binding Constants. The method offluorescence anisotropy titration as reported by Burke, T. G., Mishra,A. K., Wani, M. C. and Wall, M. E. “Lipid bilayer partitioning andstability of camptothecin drugs,” Biochemistry. 32: 5352-5364 (1993),the disclosure of which is incorporated herein by reference, wasemployed to determine the concentrations of free and bound species ofdrug in liposome suspensions containing a total drug concentration of1×10⁻⁶ M and varying lipid concentrations. All experiments wereconducted in glass tubes. The overall association constants are definedas K=[A_(B)][A_(F)][L] where [A_(B)] represents the concentration ofbound drug, [A_(F)] represents the concentration of free drug, and [L]represents the total lipid concentration of the sample.Double-reciprocal plots of the binding isotherms {1/(bound fraction ofdrug) vs. 1/[lipid]} were linear and K values were determined from theirslopes by the method of linear least squares analysis. A computerprogram based on the K=[A_(B)]/[A_(B)][L] relationship was written topredict bound drug levels for specified values of K and total drug.

Kinetics of Lactone Ring Opening of Homosilatecans, Silatecans, andCamptothecins. The hydrolysis kinetics of camptothecins in the presenceof different blood components were determined by a quantitative C18reversed-phase high-performance liquid chromatography (HPLC) assaymodified from methodologies described previously in the literature citedabove. The preparation of whole blood and fractionated blood samples wascarried out as described previously. Crystallized HSA of high purity(>97%) from Sigma Chemical (St. Louis, Mo.) was used. HSA stocksolutions were prepared in PBS buffer with a final pH of 7.40±0.05. HSAconcentrations were determined by UV absorbance at 278 nm using anextinction coefficient of 39,800 M⁻¹ cm⁻¹ (Porter, 1992). All otheragents were reagent grade and were used without further purification.High purity water provided by a Milli-Q UV PLUS purification system(Bedford, Mass.) was utilized in all experiments. HPLC solvents werefrom Fisher Scientific. Human plasma and red blood cells were obtainedfrom Central Kentucky Blood Center. Whole blood was obtained from a maledonor in heparinized tubes, stored at 5-10° C. and used as soon aspossible (typically within 1 week). Blood from mice was collected inheparinized tubes and stored at 5-10° C. until use.

HPLC assays were performed either on a Waters Alliance 2690 HPLC systemequipped with a temperature-controlled autosampler and Waters 474scanning fluorescence detector. A second HPLC system was composed of aWaters HPLC system composed of 501 HPLC pumps, 717 Plustemperature-controlled autosampler and 470 scanning fluorescencedetector. The HPLC assay procedures used for the homosilatecans aresummarized below. Solvent A consisted of acetonitrile and Solvent B was2% triethylammonium acetate, pH 5.5, with a flow rate of 1 ml/min. ForDB-38 an isocratic elution was used: 33% Solvent A; 67% Solvent B;λ_(ex):345 nm and λ_(em):518 nm. For DB-81 an isocratic elution wasused: 56% Solvent A; 44% Solvent B; and λ_(ex):375 nm and λ_(em):444 nm.For DB-90 an isocratic elution was used: 41% Solvent A; 59% Solvent B;λ_(ex):412 nm and λ_(em):526 nm. For DB-91 an isocratic elution of 42%Solvent A, 58% Solvent B, and λ_(ex):392 nm and λ_(em):562 nm.

To determine the stabilities of homosilatecans in PBS, an aliquot ofeach of the homosilatecans in DMSO was added to phosphate bufferedsaline (PBS), pH 7.4 in a HPLC autosampler vial maintained at 37° C. ina water bath to result in final drug concentration of 1 μM. Thedrug-containing vial was quickly transferred to the autosamplermaintained at 37° C. and aliquots analyzed at various time points. Allthe determinations were done in triplicate. The data was collected andanalyzed using Waters Millenium software. The fraction of lactone wascalculated using the peak area of lactone and carboxylate peak and usingthe lactone/carboxylate ratio.

To determine drug stability in whole blood, whole blood was incubated at37° C. for 30 min and pH determined. Blood pH was adjusted to 7.4+/−0.5using either 0.1 M KOH or 0.1 M HCl. Blood samples were incubated at 37°C. for 30 min and pH remeasured to ensure that it is within the rangebefore an individual assay was started. Aliquots of blood (2 ml each)were removed and placed in three disposable glass test tubes and thetubes were incubated at 37° C. An aliquot of drug in DMSO was then addedto the blood to result in a final drug concentration of 1 μM. Incubationat 37° C. was continued and 150 μl aliquots were removed at differenttime points and added to 600 μl of cold methanol (−20° C.) in aneppendorf tube. The tube was then vortexed for 10 sec and centrifuged ina table top microcentrifuge at 8000 rpm for 45 sec. The Supernatant wasremoved and placed in an autosampler vial and the vial quickly added tothe autosampler maintained at 4° C. The sample was analyzed immediatelyon the HPLC set-up. The data analysis was as described for the drug inPBS only samples.

To study the stabilities of homosilatecans in PBS containing human serumalbumin (HSA), the HSA was dissolved in PBS, pH 7.4 at the concentrationof 30 mg/ml and incubated at 37° C. The pH was measured and adjusted to7.4+/−0.5 using 0.1 M KOH or 0.1 M HCl. Incubation continued until pHstabilized within the target range. An aliquot of PBS/HSA was removedand placed in an autosampler vial and maintained at 37° C. for 10 min.An aliquot of the drug in DMSO was added to the sample resulting in afinal drug concentration of 1 μM. The vial was quickly added to the HPLCautosampler maintained at 37″C and aliquots were injected and analyzedby HPLC at different time points. The data analysis was as describedabove for the drug in PBS only samples.

To characterize the stabilities of the novel silatecans of interest inhuman plasma, frozen plasma was incubated at 37° C. in order to thaw.Blood gas was bubbled through the plasma to adjust the pH close to 7.5.Aliquots of plasma (5 ml) were incubated at 37° C. in disposable glasstest tubes and drug DMSO stock solutions added to result in a final drugconcentration of 1 μM. The samples were then allowed to incubate furtherat 37° C. Aliquots (150 μl) were removed at different time points andadded to 600 μl of cold methanol (−20° C.) in an eppendorf tube. Tubeswere vortexed for 10 sec and centrifuged in a tabletop microcentrifugeat 8000 rpm for 45 sec. The supernatant was removed and placed in anautosampler vial and the vial was quickly added to the autosamplermaintained at 4° C. The sample was analyzed by HPLC as soon as possible.The data analysis was as described for drug in PBS only samples asdescribed above. Blood gas was continuously bubbled through the plasmasamples to pH at 7.5+/−1.0.

To study the stabilities of the novel homosilatecans in the presence ofphysiologically relevant concentrations of red blood cells (RBC), thefollowing experiments were performed. Packed Red blood cells obtainedfrom the Central Kentucky Red Cross and were counted using a CoulterCell Counter. The number of cells was adjusted to 5×10¹² Cells/L usingPBS, pH 7.4 and incubated at 37° C. for 30 min. The pH of the sampleswas measured and adjusted to 7.4+/−0.5 using either 0.1 M KOH or 0.1 MHCl. RBCs were incubated at 37° C. for 30 min and the pH remeasured toensure that it was within the same range as before the assays werestarted. Aliquot of RBCs (2 ml each) were removed and placed in threedisposable glass test tubes and the tubes were incubated at 37° C.Aliquots of drug in DMSO were added to RBC suspensions in PBS to resultin final drug concentration of 1 μM. Incubation at 37° C. was continuedand 150 μl aliquots were removed at different time points and added to600 μl of cold methanol (−20° C.) present in an eppendorf tube. Thetubes were vortexed for 10 sec and centrifuged in a table topmicrocentrifuge at 8000 rpm for sec. The supernatant was removed andplaced in an autosampler vial and the vial was quickly added to theautosampler maintained at 4° C. The sample was analyzed on HPLC as soonas possible. The data analysis was as described for the drug in PBS onlysamples described above.

Fluorescence Spectral Changes Upon Homosilatecan Interactions with LipidBilayer Membranes. Fluorescence emission data were recorded forhomosilatecans in solutions of phosphate-buffered saline (PBS) at pH 7.4and ethanol. Data were also acquired for the new agents in the presenceof suspensions of small unilamellar vesicles (SUVs) composed of eitherelectroneutral dimyristoylphosphatidylcholine (DMPC) in PBS ornegatively-charged dimyristoylphosphatidylglycerol (DMPG) in PBS. Lipidconcentrations of 5 mM were used. For DB-38 all spectra were recordedusing exciting light of 410 nm at 37° C. The emission maxima for DB-38in PBS is 531 nm and this value shifts to lower values in the presenceof membranes (λ_(max) of 515 nm in the presence of DMPC vesicles andλ_(max) of 517 nm in the presence of DMPG vesicles). The spectral shiftsof DB-38 which occur in the presence of membranes indicate that theagent is capable of binding both electroneutral and negatively-chargedmembranes. Spectral recordings were initiated and completed shortlyafter the addition of the lactone form of the agent to solution orsuspension, thereby assuring that the detected signal originatespredominantly from the lactone form of the agent (and not thering-opened form).

Fluorescence emission spectra of 1 μM7-t-butyldimethylsilylhomocamptothecin (DB-81) were also examined. Lipidconcentrations of 10 mM were used. All spectra were recorded usingexciting light of 380 nm at 37° C. The emission maxima for DB-81 in PBSbuffer is 452 nm and this value shifts to lower values in the presenceof membranes (λ_(max) of 443 nm in the presence of DMPC vesicles andλ_(max) of 442 nm in the presence of DMPG vesicles). The spectral shiftsof DB-81 which occur in the presence of membranes indicate that theagent is capable of binding both electroneutral and negatively-chargedmembranes.

The fluorescence emission spectra of 1 μM7-t-butyldimethylsilyl-10-aminohomocamptothecin (DB-90) was alsoexamined. All spectra were recorded using exciting light of 430 nm at37° C. The emission maxima for DB-90 in PBS is 535 nm and this valueshifts to lower values in the presence of membranes (λ_(max) of 513 nmin the presence of DMPC vesicles and λ_(max) of 512 nm in the presenceof DMPG vesicles).

The fluorescence emission spectra of 1 μM7-t-butyldimethylsilyl-10-hydroxy-homocamptothecin (DB-91) was alsostudied. Lipid concentrations of 10 mM were used. All spectra wererecorded using exciting light of 394 nm at 37° C. The emission maximafor DB-91 in PBS is 554 nm and this value shifts to lower values in thepresence of membranes (λ_(max) of 441 nm in the presence of DMPCvesicles and λ_(max) of 434 nm in the presence of DMPG vesicles

The fluorescence emission spectra of 1 μM of the carboxylate form of7-t-butyl-dimethylsilyl-10-aminohomocamptothecin (DB90 carboxylate) wasalso studied. Lipid concentrations of 0.15 mM were used. Theconcentration of lipid used in these experiments was greater than in theexperiments conducted using the corresponding lactone form of DB-90; thehigher lipid concentrations were used because of the reduced membraneassociations of the opened-ring form of the drug relative to theclosed-ring lactone form of the drug. All spectra were recorded usingexciting light of 430 nm at 37° C. The emission maxima for DB-90carboxylate in PBS is 529 nm and this value shifts to lower values inthe presence of membranes (λ_(max) of 512 nm in the presence of DMPCvesicles and λ_(max) of 512 nm in the presence of DMPG vesicles

The fluorescence emission spectra of 1 μM of the ring-opened orcarboxylate form of 7-t-butyldimethylsilyl-10-hydroxyhomocamptothecin(DB-91 carboxylate) were also acquired. Lipid concentrations of 0.15 Mwere used (the higher concentration of lipid required in theseexperiments to promote binding was necessitated by the reduced membraneassociations of the opened-ring form of the agent relative to theclosed-ring lactone form of the agent. The emission maxima for DB-91carboxylate in PBS is 549 nm and this value shifts to lower values(λ_(max) of 450 nm in the presence of DMPC vesicles and λ_(max) of 446nm in the presence of DMPG vesicles).

Normalized fluorescence emission spectra of 1 μM of the lactone versuscarboxylate forms of 7-t-butyldimethylsilyl-10-aminohomocamptothecin(DB-90 and DB-90 carboxylate, respectively) in PBS at 37° C. werestudied. Fluorescence emission spectral data indicate that upon ringopening there is a slight shifting of the spectra to the shorterwavelength region (or shifting of the spectra more towards the blueregion of light). The two spectra were recorded using exciting light of402 nm.

Normalized fluorescence emission spectra of 1 μM of the lactone versuscarboxylate forms of 7-t-butyldimethylsilyl-10-hydroxylhomocamptothecin(DB-91 and DB-91 carboxylate, respectively) in PBS at 37° C. wereacquired. Fluorescence emission spectral data indicate that upon ringopening there is a slight shifting of the spectra to the shorterwavelength region (or shifting of the spectra more towards the blueregion of light). The two spectra were recorded at 394 nm.

Direct Observation of Fluorescence Spectral Changes Upon HomosilatecanPartitioning in Red Blood Cells. Spectral recordings were initiated andcompleted shortly after the addition of the lactone form of the agent tosolution, thereby assuring that the detected signal originatespredominantly from the lactone form of the agent (and not thering-opened form). The emission maxima for DB-91 in PBS is 554 nm butthis value shifts significantly to a λ_(max) of approximately 410 nm inanhydrous ethanol. Because DB-91 contains a 10-hydroxy functionality,the possibility exists that fluorescence can occur from two distinctspecies. In an aprotic solvent or non-aqueous microenvironment aprotonated (with respect to the 10-hydroxy functionality) speciespredominates, while in protic solvents such as water a deprotonatedexcited-state complex predominates. The 554 nm peak is correlated withthe deprotonated excited-state complex while the λ_(max) ofapproximately 410 nm correlates with the protonated excited-statecomplex. The formation of the deprotonated excited-state complex isgreatly facilitated by the presence of water; even at small amounts ofwater such as 1% a peak is apparent around 550 nm which correlates withthe water-facilitated formation of the deprotonated excited-statecomplex. In FIGS. 6 and 7 we study the extent of protonatedexcited-state complex formation and use this parameter as a relativemeasure of lipophilicity for two 7-modified campotothecins (DB-91 andSN-38) with each containing the 10-hydroxy functionality. Spectralrecordings were initiated and completed shortly after the addition ofthe lactone form of the agent to solution, thereby assuring that thedetected signal originates predominantly from the lactone form of theagent (and not the ring-opened form). The emission maxima for DB-91 inPBS is 554 nm. In the presence of red blood cells, a peak with asignificantly lower λ_(max) value is observed indicating that the agentis capable of partitioning into the red blood cell membranes. Themembranes of the red blood cells provide a hydrophobic microenvironmentfrom which the protonated excited-state complexes can form and fluorescefrom. Comparison of the emission spectra of DB-91 in the presence ofhuman erythrocytes with that of clinically relevant7-ethyl-10-hydroxycamptothecin (FIG. 7) indicate there is more extensiveprotonated excited-state complex formation in the case of DB-91. Thesefindings corroborate model membrane studies indicating the membranebinding of SN-38 is significantly less than the extensive interactionsnoted for DB-91 (SN-38 displays a K_(DMPC) value of 300 M⁻¹ whereasDB-91 displays a K_(DMPC) value of 8,000 M⁻¹). The novel homosilatecanDB-91 is a more lipophilic, erythrocyte-interactive agent relative tothe known compound 7-ethyl-10-hydroxycamptothecin (SN-38). The emissionmaxima for SN-38 in PBS is approximately 550 nm. The SN-38 agent, likeDB-91, also contains a 10-hydroxy functionality and, as a consequence,SN-38 also displays fluorescence spectral characteristics which aresensitive to the presence of water. In the presence of red blood cells,a peak with a significantly lower λ_(max) value (approximately 440 nm)is observed for SN-38 indicating that the agent is capable ofpartitioning into the red blood cell membranes. However, the peak at thelower λ_(max) value is reduced for SN-38 relative to the situationobserved for DB-91 (see FIG. 6). These results indicate there is moreextensive protonated excited-state complex formation in the case forDB-91, corroborating that the novel homosilatecan DB-91 is a morelipophilic, erythrocyte-interactive agent relative to the known compound7-ethyl-10-hydroxycamptothecin (SN-38).

Anticancer Activities of Homosilatecans as Determined By In Vitro CellCulture Experiments. Cytotoxicity measurements were conducted usingMDA-MB-435 tumorigenic human breast cancer cells. The cells were exposedto a range of drug concentrations for 72 hr exposure periods and thenviability was assessed using a sulphorrhodamine B (SRB) assay. The SRBmeasures the total protein levels in the living cells. Proteins fromdead cells are lysed and removed in the washing step before TCAfixation. However, it is possible that cells in the early stage of deathstill have their membrane integrity and therefore retain the proteincontents inside. As a result, the optical density at 490 nm cansometimes be overestimated and the cytotoxicity underestimated. Tovalidate the SRB assay, a diverse range of chemotherapeutic agents havebeen tested across multiple panels of tumor cell lines, and closecorrelations have been found with standard tetrazolium (MIT) assay andclonogenic assays. The SRB assay is now a well regarded assay and wasrecently approved by NCI as a standard assay for anticancer drugscreening. Using the SRB assay, the cytotoxicity values for cellsexposed to our novel homosilatecans for 72 hrs. were determined.Cytotoxicities of homosilatecans and camptothecin against MDA-MB-435tumorigenic metastatic human breast cancer cells in the absence andpresence of human serum albumin were determined and are summarized inTable 5. Each IC₅₀ value represents the average of three separate trialswith each dosage level studied in triplicate.

(+/−)4-Ethyl-8-methoxy-6-trimethylsilanyl-3,4-dihydro-1H-pyrano[3,4-c]pyridine-3,4-diol(4)

To a round bottom flask was added N-Methylmorpholine N-oxide (0.89 g,7.6 mmol) followed by H₂O (10 mL) and t-BuOH (10 mL). A 2.5 weightpercent solution of OsO4 in t-BuOH (0.5 mL) was added followed by enolether (3) (0.5 g, 1.9 mmol). After 12 hours, at 22° C., Na₂SO₃ (1.0 g)was added to the mixture. After 30 minutes the mixture was diluted withH₂O (100 mL) and extracted with CH₂Cl₂ (2×100 mL). The organic layer wasdried (MgSO₄) and the crude residue was chromatographed (hexanes:EtOAc3:1) to yield lactol (4) 0.55 g (98%) as a white solid: IR (CHCl₃, cm⁻¹)3602, 3569, 3398, 3022, 2950, 1579, 1456, 1351; ¹H NMR (300 MHz, CDCl₃)δ 0.22 (s, 9H), 0.83 (t, J=7 Hz, 3H), 1.71-1.79 (m, J=7 Hz, 2H), 2.91(s, 1H), 3.90 (s, 3H), 4.19 (d, J=5 Hz, 1H), 4.53 (d, J=16 Hz, 1H), 4.70(d, J=16 Hz, 1H), 5.06 (d, J=5 Hz, 1H), 7.25 (s, 1H); ¹³C NMR (75 MHz,CDCl₃) δ −1.7, 7.8, 31.5, 53.1, 58.8, 70.9, 93.8, 115.1, 119.7, 145.8,158.0, 162.8; HRMS (EI) m/z calcd for C₁₄H₂₃NO₄Si (M⁺) 473.0519. found473.0507 LRMS (EI) m/z 473 (M⁺), 458, 386, 360, 346, 139, 73, 57.

Formic acid 2-methoxy-4-propionyl-6-trimethylsilanyl-pyridin-3-yl methylester (5)

To a round bottom flask was added lactol (4) (0.100 g, 0.34 mmol)followed by AcOH (9 mL) and lead tetraacetate (0.18 g, 0.406 mmol).After 3 hours at 50° C. the mixture was poured into ice cold sat. NaHCO₃and extracted with ether (3×75 mL). The organic layer was dried (MgSO₄)and chromatographed (hexanes:EtOAc 95:5) to yield keto-formate ester (5)91 mg (91%) as a clear oil: IR (neat, cm⁻¹) 2963, 2902, 1733, 1556,1455, 1345; ¹H NMR (300 MHz, CDCl₃) δ 0.30 (s, 9H), 1.21 (t, J=7 Hz,3H), 2.75-2.95 (m, J=7 Hz, 2H), 4.02 (s, 3H), 5.28 (s, 2H), 7.07 (s,1H), 8.05 (s, 1H); ¹³C NMR (75 MHz, CDCl₃) δ −1.8, 7.9, 35.9, 54.0,57.6, 112.9, 118.7, 148.5, 160.8, 162.2, 167.6, 205.6; HRMS (EI) m/zcalcd for C₁₄H₂₁NO₄Si (M⁺) 295.1240. found 295.1239 LRMS (EI) m/z 295(M⁺), 280, 267, 250, 234, 222, 206, 176, 162, 103, 89, 79, 73, 57.

(+/−)3-Hydroxy-3-(3-hydroxymethyl-2-methoxy-6-trimethylsilanyl-pyridin-4-yl)-pentanoicacid tert-butyl ester (6)

To a flame dried flask was added keto-formate ester (5) (0.5 g, 1.69mmol) followed by dioxane (20 mL). α-Bromo-tert-butylacetate (0.9 mL,6.08 mmol) was added followed by activated Zn (0.59 g, 9.1 mmol). The Znwas activated by the Cava method as set forth in the J. Organic. Chem.,47, p. 5030 (1982), the disclosure of which is incorporated herein byreference. Next I₂ (0.16 g, 0.63 mmol) was added and the mixture wassonicated for 3.2 hours. After sonication the mixture was diluted withH₂O (100 mL) and ether (100 mL). The resulting emulsion was filteredthrough a pad of celite, the phases were separated and the aqueous layerwas extracted with ether (2×100 mL). The combined ether extracts weredried (MgSO₄) and chromatographed (hexanes:EtOAc 85:15) to yieldbeta-hydroxy ester (6) 0.50 g (78%) as a clear oil: IR (neat, cm⁻¹)3469, 2980, 1705, 1575, 1545, 1447, 1342, 1248, 1153; ¹H NMR (300 MHz,C₆D₆) 60.38 (s, 9H), 0.79 (t, J=7 Hz, 3H), 1.15 (s, 9H), 1.75-1.92 (m,J=7 Hz, 2H), 2.52 (d, J=16 Hz, 1H), 2.79 (d, J=16 Hz, 1H), 3.03 (t, J=7Hz, 2H), 3.74 (s, 3H), 5.18 (d, J=7 Hz, 2H), 5.19 (s, 1H), 7.18 (s, 1H);¹³C NMR (75 MHz, C_(E)D_(E)) δ −1.9, 8.1, 27.6, 35.5, 45.6, 53.0, 57.0,77.3, 81.5, 120.9, 121.8, 152.3, 162.6, 163.3, 172.3; HRMS (EI) m/zcalcd for C₁₉H₃₁NO₄Si (M-H₂O) 365.2022. found 365.2028 LRMS (EI) m/z 383(M⁺), 365, 336, 309, 280, 262, 250, 208, 89, 73, 57.

(+/−)5-Ethyl-4,5-dihydro-5-hydroxy-7-trimethylsilyl-9-methoxyoxepino[3,4-c]pyridin-3(1H)-one(7)

To a 100 mL flask was added the beta hydroxy ester (6) (0.75 g, 1.9mmol) followed by trifluoroacetic acid (150 mL). After 24 hours, themixture was poured into sat. NaHCO₃ (pH 8) and extracted with ether(3×100 mL). The organic phase was dried (MgSO₄) and chromatographed(hexanes:EtOAc 2:1) to give beta-hydroxy lactone (7) 0.48 g (79%) as awhite solid: IR (CHCl₃, cm⁻¹) 3020, 2978, 2873, 1742, 1561, 1448, 1384,1348, 1110, 909, 842; ¹H NMR (300 MHz, CDCl₃) δ 0.22 (s, 9H), 0.83 (t,J=7 Hz, 3H), 1.81-1.88 (m, J=7 Hz, 2H), 1.37 (br s, 1H), 3.00 (d, J=14Hz, 1H), 3.32 (d, J=14 Hz, 1H), 3.91 (s, 3H), 5.18. (d, J=15 Hz, 1H),5.42 (d, J=15 Hz, 1H), 7.26 (s, 1H); ¹³C NMR (75 MHz, CDCl₃) δ −1.9,8.4, 33.9, 42.9, 53.8, 62.2, 73.8, 114.7, 121.2, 151.6, 159.8, 165.9,172.3; HRMS (EI) m/z calcd for C₁₅H₂₃NO₄Si (M⁺) 309.1396. found 309.1399LRMS (EI) m/z 309 (M⁺), 294, 266, 252, 238, 89.

(+/−)5-Ethyl-4,5-dihydro-5-hydroxy-7-iodo-9-methoxyoxepino[3,4-c]pyridin-3(1H)-one(8)

To a flame dried flask at 0° C. was added beta hydroxy lactone (7) (0.94g, 3.0 mmol) followed by dry CH₂Cl₂ (25 mL). ICl (3.2 g, 19.7 mmol), wasadded to a flame dried flask at −78° C. The flask was taken out of thebath, warmed slightly, excess moisture was wiped from the outside and itwas quickly weighed under nitrogen. After weighing it was returned tothe −78° C. bath and diluted with ice cold CCl₄ (16 mL) to give a 1.2 Msolution of ICl. The resulting ICl solution was transferred to an icebath and allowed to equilibrate to 0° C. A portion of the ICl solution(10.1 mL) was transferred to the mixture dropwise in the dark. Afterhours in the dark, the mixture was poured into a 1:1 solution (100 mL)of 5% Na₂SO₃ and sat. Brine and extracted with EtOAc (3×100 mL). Theorganic layer was dried (MgSO₄) and chromatographed (hexanes:EtOAc 3:1)to give beta hydroxy lactone (7) 0.43 g (46%) and iodolactone (8) 0.41 g(37%): IR (CHCl₃, cm⁻¹) 2974, 2951, 1747, 1573, 1554, 1359, 1278, 1212,1054, 870; ¹H NMR (300 MHz, CDCl₃) δ 0.84 (t, J=7 Hz, 3H), 1.78-1.85 (m,J=7 Hz, 2H), 2.98 (br d, J=14 Hz, 2H), 3.30 (d, J=14 Hz, 1H), 3.90 (s,3H), 5.10 (d, J=15 Hz, 1H), 5.35 (d, J=15 Hz, 1H), 7.51 (s, 1H); ¹³C NMR(75 MHz, CDCl₃) δ 8.4, 37.4, 42.7, 55.0, 61.8, 73.4, 114.0, 114.9,127.3, 155.3, 159.8, 171.9; HRMS (EI) m/z calcd for C₁₂H₁₄INO₄ (M⁺)362.9967. found 362.9955 LRMS (EI) m/z 363 (M⁺), 334, 326, 317, 302,292, 262, 234, 162, 137, 120, 57.

(+/−)5-Ethyl-1,4,5,8-tetrahydro-5-hydroxy-7-iodooxepino[3,4-c]pyridine-3,9-dione(9)

To a flame dried flask was added iodolactone (8) (0.33 g, 0.90 mmol)followed by dry acetonitrile (12 mL). Sodium iodide (0.22 g, 1.44 mmol)was added followed by chlorotrimethylsilane (0.18 mL, 1.44 mmol). Theresulting mixture was stirred at 22° C. for 15 minutes at which pointH₂O (7.6 μL, 0.42 mmol) was added and the mixture was heated at 60° C.After 5 hours at 60° C. the mixture was poured into a 1:1 solution of 5%Na₂SO₃/Brine (75 mL) and then quickly extracted with EtOAc (6×75 mL).The organic layer was dried (MgSO₄) and chromatographed (CH₂Cl₂:MeOH95:5) to yield iodopyridone (9) as a white solid 0.19 g (61%): ¹H NMR(300 MHz, CDCl₃/CD₃OD) δ 0.62 (t, J=7 Hz, 3H), 1.45-1.54 (m, J=7 Hz,2H), 2.80 (d, J=14 Hz, 1H), 2.97 (d, J=14 Hz, 1H), 4.93 (d, J=15 Hz,1H), 5.06 (d, J=15 Hz, 1H), 6.66 (s, 1H); ¹³C NMR (75 MHz, CDCl₃) δ 7.5,35.6, 41.9, 61.6, 72.5, 94.4, 118.3, 121.1, 156.5, 162.6, 172.7; HRMS(EI) m/z calcd for C₁₁H₁₂INO₄ (M⁺) 348.9811. found 348.9815 LRMS (EI)m/z 349 (M⁺), 331, 320, 303, 289, 278, 264, 250, 162, 150, 122, 94, 57.

Preparation of N-Alkylated Iodopyridones (+/−)5-Ethyl-1,4,5-trihydro-5-hydroxy-7-iodo-8-(3-trimethylsilyl-2-propynyl)-oxepino[3,4-c]pyridine-3,9-dione(10a)

To a flame dried flask was added iodopyridone (9) (0.16 g, 0.46 mmol)followed by dry DME (3.8 mL) and DMF (0.95 mL). This solution waslowered to 0° C. and NaH, 60% dispersion in oil, (19.3 mg, 0.483 mmol)was added portionwise. After 15 minutes 2 eq of vacuum flame dried LiBr(81 mg, 0.92 mmol) was added and the mixture was raised to 22° C. After25 minutes at 22° C., the trimethylsilylpropargyl bromide (0.130 mL,0.92 mmol) was added and the mixture was heated at 65° C. After 16hours, the mixture was poured into brine (50 mL) and extracted withEtOAc (8×30 mL). The EtOAc layer was dried (MgSO₄) and chromatographed(CH₂Cl₂:EtOAc 80:20) to give the desired N alkylated pyridone (10a) 134mg (63%) as a white foam: ¹H NMR (300 MHz, CDCl₃) δ 0.005 (s, 9H), 0.80(t, J=7 Hz, 3 H), 1.60-1.74 (m, J=7 Hz, 2H), 2.94 (d, J=14 Hz, 1H), 3.11(d, J=14 Hz, 1H), 3.60 (br s, 1H), 4.82 (d, J=17 Hz, 1H), 5.01 (d, J=17Hz, 1H), 5.09 (d, J=15 Hz, 1H), 5.26 (d, J=15 Hz, 1H), 7.01 (s, 1H); ¹³CNMR (75 MHz, CDCl₃/CD₃OD) δ −0.44, 7.9, 35.7, 42.2, 45.2, 62.3, 72.6,90.7, 98.1, 99.2, 119.9, 122.3, 155.1, 160.6, 172.6; HRMS (EI) m/z calcdfor C₁₇H₂₂INO₄Si (M⁺) 459.0363. found 459.0366 LRMS (EI) m/z 459 (M⁺),444, 388, 306, 111, 96, 83, 73, 57.

(+/−)5-Ethyl-1,4,5-trihydro-5-hydroxy-7-iodo-8-(3-tert-butyldimethylsilyl-2-propynyl)-oxepino[3,4-c]pyridine-3,9-dione(10b)

Following the procedure outlined above iodopyridone (7) (0.16 g, 0.46mmol) was N alkylated with the TBDMS propargyl bromide (0.21 g, 0.92mmol). Flash chromatography (CH₂Cl₂:EtOAc 9:1) gave iodopyridone (8b)134 mg (58%) as a white foam: ¹H NMR (300 MHz, CDCl₃) δ 0.097 (s, 6H),0.92 (br s, 12H), 1.82-1.89 (br m, 2H), 3.01 (d, J=14 Hz, 1H), 3.33 (d,J=14 Hz, 1H), 3.48 (br s, 1H), 5.07 (s, 2H), 5.12 (d, J=15 Hz, 1H), 5.47(d, J=15 Hz, 1H), 7.10 (s, 1H); ¹³C NMR (75 MHz, CDCl₃) δ −4.7, 8.3,16.6, 26.3, 36.0, 42.6, 45.3, 62.8, 73.5, 89.4, 98.6, 99.5, 119.5,122.9, 154.1, 160.4, 172.0; HRMS (EI) m/z calcd for C₂₀H₂₈INO₄Si (M⁺)501.0832. found 501.0843 LRMS (EI) m/z 501 (M⁺), 444, 402, 335, 318,169, 121, 96, 57.

(+/−)5-Ethyl-1,4,5,13-tetrahydro-5-hydroxy-12-trimethylsilyl-3H,15H-oxepino[3′,4′:6,7]indolizino[1,2-b]quinoline-3,15-dione(1 h) (7-trimethylsilylhomocamptothecin)

To an oven dried pressure tube under Ar was added the iodopyridone (10a)(15 mg, 0.033 mmol) followed by benzene (0.25 mL) and t-BuOH (0.5 mL).Next phenylisonitrile (13.6 mg, 0.13 mmol) and hexamethylditin (16.0 mg,0.049 mmol) were added and the tube was flushed with Ar, sealed andplaced in front of a 275W GE sunlamp. After 12 hours of irradiation, themixture was concentrated and chromatographed (CH₂Cl₂:acetone 5:1) toyield homocamptothecin (1h) 5.2 mg (36%) as a tan solid: ¹H NMR (300MHz, CDCl₃/CD₃OD) δ 0.64 (s, 9H), 0.96 (t, J=7 Hz, 3 H), 1.96-2.05 (m,J=7 Hz, 2H), 3.19 (d, J=14 Hz, 2H), 3.46 (d, J=14 Hz, 1H), 5.34 (s, 2H),5.44 (d, J=15 Hz, 1H), 5.63 (d, J=15 Hz, 1H), 7.65-7.71 (m, 2H),7.78-7.84 (m, 1H), 8.18 (d, J=8 Hz, 1H), 8.27 (d, J=8 Hz, 1H); HRMS (EI)m/z calcd for C₂₄H₂₆N₂O₄Si (M⁺) 434.1662. found 434.1679 LRMS (EI) m/z434 (M⁺), 419, 388, 374, 363, 347, 335, 320, 303, 289, 275, 261, 247,231, 219, 174, 149, 73.

(+/−) 5-Ethyl-1,4,5,13-tetrahydro-5-hydroxy-10-(tert-butyloxycarbonylamino)-12-trimethylsilyl-3H,15H-oxepino[3′,4′:6,7]indolizino[1,2-b]quinoline-3,15-dione(1c) (10-tert-butyloxycarbonylamino-7-trimethylsilylhomocamptothecin)

Following the procedure described above, iodopyridone (10a) (30 mg,0.065 mmol) was reacted with para-bocaminophenylisonitrile (57 mg, 0.26mmol) and hexamethylditin (32.2 mg, 0.1 mmol) in benzene (0.5 mL) andt-BuOH (1 mL). Chromatography (CH₂Cl₂:Acetone 7:1) gave compound (1c)18.8 mg (53%) as a brown solid: IR (CHCl₃, cm⁻¹) 3022, 3007, 1736, 1655,1594, 1528, 1155, 1062; ¹H NMR (500 MHz, CDCl₃) δ 0.70 (s, 9H), 0.96 (t,J=7 Hz, 3 H), 1.61 (s, 9H), 1.85-2.10 (m, J=7 Hz, 2H), 3.31 (d, J=13 Hz,1H), 3.41 (d, J=13 Hz, 1H), 5.11 (d, J=19 Hz, 1H), 5.34-5.41 (m, 2H),5.61 (d, J=15 Hz, 1H), 6.96 (s, 1H), 7.19-7.40 (m, 1H), 7.62 (s, 1H),8.37 (s, 1H); ¹³C NMR (125 MHz, CDCl₃) δ 1.43, 8.3, 28.4, 35.8, 42.6,52.4, 62.3, 73.9, 81.2, 100.3, 115.1, 122.2, 123.0, 130.7, 132.6, 134.8,137.1, 143.4, 143.6, 145.1, 148.2, 152.6, 156.5, 159.8, 171.8; HRMS (EI)m/z calcd for C₂₉H₃₅N₃O₆Si (M⁺) 549.2295. found 549.2274 LRMS (EI) m/z549 (M⁺), 493, 475, 449, 433, 415, 404, 389, 378, 350, 304, 260, 195,182, 73.

(+/−)5-Ethyl-1,4,5,13-tetrahydro-5-hydroxy-10-acetoxy-12-trimethylsilyl-3H,15H-oxepino[3′,4′:6,7]indolizino[1,2-b]quinoline-3,15-dione(10-acetoxy-7-trimethylsilylhomocamptothecin)

Following the procedure described above, iodopyridone (10a) (30 mg,0.065 mmol) was reacted with para-acetoxyphenylisonitrile (42 mg, 0.26mmol) and hexamethylditin (32.2 mg, 0.1 mmol) in benzene (0.5 mL) andt-BuOH (1 mL). Chromatography (CH₂Cl₂:Acetone 5:1) gave the product 6.6mg (21%) as a tan solid: IR (CHCl₃, cm⁻¹) 3025, 2992, 2953, 1753, 1657,1600, 1504, 1193; ¹H NMR (300 MHz, CDCl₃) δ 0.67 (s, 9H), 0.98 (t, J=7Hz, 3H), 1.99-2.07 (m, 2H), 2.42 (s, 3H), 3.26 (d, J=14 Hz, 1H), 3.45(d, J=14 Hz, 1H), 3.66 (br s, 1H), 5.18 (d, J=19 Hz, 1H), 5.35 (d, J=15Hz, 1H), 5.39 (d, J=19 Hz, 1H), 5.66 (d, J=15 Hz, 1H), 7.38 (dd, J₁=9Hz, J₂=2 Hz, 1H), 7.40 (s, 1H), 7.88 (d, J=9 Hz, 1H), 7.92 (d, J=2 Hz,1H); ¹³C NMR (125 MHz, CDCl₃) δ 1.6, 8.3, 21.5, 35.9, 42.6, 52.2, 62.2,74.0, 100.5, 118.9, 122.9, 124.7, 131.3, 132.2, 135.0, 144.1, 144.8,145.0, 148.9, 150.0, 156.0, 159.7, 169.1, 171.5; HRMS (EI) m/z calcd forC₂₆H₂₈N₂O₆Si (M⁺) 492.1717. found 492.1707 LRMS (EI) m/z 492 (M⁺), 477,459, 450, 432, 421, 403, 393, 379, 365, 351, 336, 147.

(+/−)5-Ethyl-1,4,5,13-tetrahydro-5-hydroxy-12-tert-butyldimethylsilyl-3H,15H-oxepino[3′,4′:6,7]indolizino[1,2-b]quinoline-3,15-dione(1g) (7-tert-butyldimethylsilylhomocamptothecin)

Following the procedure described above, iodopyridone (10b) (25 mg, 0.05mmol) was reacted with phenylisonitrile (15.5 mg, 0.15 mmol) andhexamethylditin (25 mg, 0.075 mmol) in benzene (0.75 mL). Chromatography(CH₂Cl₂:Acetone 7:1) gave compound (1g) 6.4 mg (27%) as a tan solid: IR(CHCl₃, cm⁻¹) 3027, 2958, 2932, 2859, 1745, 1655, 1600, 1269, 1065; ¹HNMR (300 MHz, CDCl₃) δ 0.69 (s, 3H), 0.70 (s, 3H), 0.92 (t, J=7 Hz, 3H),1.00 (s, 9H), 1.92-2.02 (m, J=7 Hz, 2H), 3.23 (d, J=13 Hz, 1H), 3.39 (d,J=13 Hz, 1H), 3.90 (br s, 1H), 5.11 (d, J=19 Hz, 1H), 5.31 (d, J=15 Hz,1H), 5.40 (d, J=19 Hz, 1H), 5.60 (d, J=15 Hz, 1H), 7.35 (s, 1H),7.39-7.49 (m, 2H), 7.70 (d, J=8 Hz, 1H), 8.07 (d, J=8 Hz, 1H); ¹³C NMR(75 MHz, CDCl₃) δ −0.5, −0.3, 8.3, 19.4, 27.3, 35.9, 42.7, 52.9, 62.3,74.0, 100.3, 122.8, 126.9, 129.4, 130.3, 132.7, 136.0, 143.3, 145.3,147.6, 150.1, 156.1, 159.9, 171.5; HRMS (EI) m/z calcd for C₂₇H₃₂N₂O₄Si(M⁺) 476.2131. found 476.2118 LRMS (EI) m/z 476 (M⁺), 458, 430, 419,405, 389, 377, 361, 345, 319, 304, 275, 149, 117, 91, 73, 56.

(+/−) 5-Ethyl-1,4,5,13-tetrahydro-5-hydroxy-10-(tert-butyloxycarbonylamino)-12-tert-butyldimethylsilyl-3H,15H-oxepino[3′,4′:6,7]indolizino[1,2-b]quinoline-3,15-dione(1a)(10-tert-butyloxycarbonylamino-7-tert-butyldimethylsilylhomocamptothecin)

Following the procedure described above, iodopyridone (10b) (45 mg,0.089 mmol) was reacted with para-bocaminophenylisonitrile (58 mg, 0.27mmol) and hexamethylditin (45 mg, 0.13 mmol) in benzene (1.3 mL).Chromatography (CH₂Cl₂:Acetone 10:1) gave compound (1a) 7.8 mg (15%) asa tan solid: IR (CHCl₃, cm⁻¹) 3435, 3022, 2931, 2859, 1738, 1654, 1563,1528, 1156; ¹H NMR (300 MHz, CDCl₃) δ 0.76 (s, 3H), 0.77 (s, 3H), 0.96(t, J=7 Hz, 3H), 1.10 (s, 9H), 1.62 (s, 9H), 1.91-2.07 (m, J=7 Hz, 2H),3.34 (d, J=14 Hz, 1H), 3.41 (d, J=14 Hz, 1H), 4.42 (br s, 1H), 5.09 (d,J=19 Hz, 1H), 5.38 (d, J=15 Hz, 1H), 5.47 (d, J=19 Hz, 1H), 5.62 (d,J=15 Hz, 1H), 6.99 (br s, 1H), 7.21-7.25 (m, 2H), 7.45 (d, J=9 Hz, 1H),8.37 (d, J=2 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃) δ −0.9, −0.5, 8.3, 19.6,27.4, 28.4, 35.5, 42.7, 53.0, 62.2, 73.8, 81.1, 100.0, 116.2, 122.2,123.0, 130.3, 133.5, 136.3, 136.9, 144.4, 145.2, 148.2, 152.6, 156.4,160.0, 171.5; HRMS (EI) m/z calcd for C₃₂H₄₁N₃O₆Si (M⁺) 591.2765. found591.2751 LRMS (EI) m/z 534 (M-57), 516, 488, 477, 459, 435, 417, 393,375, 111, 97, 83, 69, 57.

(+/−)5-Ethyl-1,4,5,13-tetrahydro-5-hydroxy-10-acetoxy-12-tert-butyldimethylsilyl-3H,15H-oxepino[3′,4′:6,7]indolizino[1,2-b]quinoline-3,15-dione(1e) (10-acetoxy-7-tert-butyldimethylsilylhomocamptothecin)

Following the procedure described above, iodopyridone (10b) (45 mg,0.089 mmol) was reacted with para-acetoxyphenylisonitrile (43 mg, 0.27mmol) and hexamethylditin (45 mg, 0.13 mmol) in benzene (1.3 mL).Chromatography (CH₂Cl₂:Acetone 10:1) gave compound (1e) 9.6 mg (20%) asa tan solid: ¹H NMR (300 MHz, CDCl₃) δ 0.73 (s, 3H), 0.74 (s, 3H), 0.97(t, J=7 Hz, 3H), 1.07 (s, 9H), 1.94-2.08 (m, J=7 Hz, 2H), 2.42 (s, 3H),3.29 (d, J=14 Hz, 1H), 3.44 (d, J=14 Hz, 1H), 4.05 (br s, 1 H), 5.16 (d,J=19 Hz, 1H), 5.37 (d, J=15 Hz, 1H), 5.48 (d, J=19 Hz, 1H), 5.65 (d,J=15 Hz, 1H), 7.31 (dd, J₁=9 Hz, J₂=2 Hz, 1H), 7.36 (s, 1H), 7.70 (d,J=9 Hz, 1H), 7.97 (d, J=2 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃) δ −0.7,−0.5, 8.3, 19.3, 21.5, 27.2, 35.8, 42.7, 52.9, 62.2, 73.9, 100.4, 120.1,122.8, 124.7, 131.1, 133.0, 136.5, 143.0, 145.0, 145.4, 148.9, 149.9,156.2, 159.9, 169.0, 171.5; HRMS (EI) m/z calcd for C₂₉H₃₄N₂O₆Si (M⁺)534.2186. found 534.2188 LRMS (EI) m/z 534 (M⁺), 516, 488, 477, 459,435, 417, 393, 375, 335, 320, 291, 275, 234, 164, 137, 125, 111, 97, 83,69, 57.

(+/−)5-Ethyl-1,4,5,13-tetrahydro-5-hydroxy-10-hydroxy-12-tert-butyldimethylsilyl-3H,15H-oxepino[3′,4′:6,7]indolizino[1,2-b]quinoline-3,15-dione(1f) (10-hydroxy-7-tert-butyldimethylsilylhomocamptothecin)

Compound (1e) (11.9 mg, 0.022 mmol) was dissolved in H₂O (0.3 mL) andMeOH (0.3 mL). Next K₂CO₃ (7.5 mg, 0.054 mmol) was added and the mixturewas stirred at 22° C. After 4 h the solvent was evaporated and theresidue was dissolved in CH₂Cl₂ (2 mL) and TFA (2 mL). After stirring at22° C. for 16 h sat. NaHCO₃ was carefully added until pH 5 was attained.At this point the solution was extracted with EtOAc (3×10 mL) and theorganic layer was dried (Na₂SO₄) and concentrated. The residue waschromatographed twice (1:CH₂Cl₂:MeOH:AcOH 94:5:1) (2:CH₂Cl₂:Acetone 5:1)to give compound (1f) 8.6 mg (79%) as a yellow solid: ¹H NMR (300 MHz,CDCl₃/CD₃OD) δ 0.65 (s, 6H), 0.90-0.99 (m, 12H), 1.89-2.05 (m, 2H), 3.14(d, J=14 Hz, 1H), 3.34 (d, J=14 Hz, 1H), 5.23 (s, 2H), 5.35 (d, J=15 Hz,1H), 5.57 (d, J=15 Hz, 1H), 7.42 (dd, J₁=9 Hz, J₂=2 Hz, 1H), 7.58 (d,J=2 Hz, 1H), 7.70 (s, 1H), 8.16 (d, J=9 Hz, 1H); ¹³C NMR (125 MHz,CDCl₃/CD₃OD) δ −1.1, 8.1, 19.2, 26.9, 36.1, 42.1, 52.8, 62.1, 73.5,101.8, 111.5, 122.7, 123.6, 127.5, 129.0, 135.0, 136.6, 139.8, 143.1,145.5, 156.7, 156.9, 159.6, 172.8; HRMS (EI) m/z calcd for C₂₇H₃₂N₂O₅Si(M⁺) 492.2080. found 492.2087 LRMS (EI) m/z 492 (M⁺), 474, 446, 435,421, 393, 375, 346, 335, 315, 291, 273, 259, 231, 183, 155.

(+/−)5-Ethyl-1,4,5,13-tetrahydro-5-hydroxy-10-amino-12-tert-butyldimethylsilyl-3H,15H-oxepino[3′,4′:6,7]indolizino[1,2-b]quinoline-3,15-dione(1b) (10-amino-7-tert-butyldimethylsilyhomocamptothecin)

Trifluoroacetic acid (0.1 mL) was added to a solution containing CH₂Cl₂(0.5 mL) and compound (1a) (8.1 mg, 0.014 mmol) and the contents werestirred at 22° C. After 5h the mixture was poured into sat. NaHCO₃ (2mL) and extracted with EtOAc (6×2 mL). The EtOAc was dried (Na₂SO₄),concentrated and chromatographed (CH₂Cl₂:MeOH 96:4) to give (1b) 6 mg(89%) as a yellow solid: ¹H NMR (300 MHz, CDCl₃/CD₃OD) δ 0.28 (s, 6H),0.78-0.88 (m, 12H), 1.78-1.90 (m, 2H), 3.04 (d, J=14 Hz, 1H), 3.24 (d,J=14 Hz, 1H), 5.02-5.11 (m, 2H), 5.24 (d, J=15 Hz, 1H), 5.46 (d, J=15Hz, 1H), 7.20 (s, 1H), 7.26 (dd, J₁=⁹ Hz, J₂=2 Hz, 1H); ¹³C NMR (125MHz, CDCl₃/CD₃OD) δ −1.0, 8.0, 19.1, 26.9, 36.1, 42.1, 52.7, 62.1, 73.4,100.7, 122.0, 123.2, 130.5, 134.3, 136.8, 141.8, 144.2, 147.1, 156.7,159.7, 172.8; HRMS (EI) m/z calcd for C₂₇H₃₃N₃O₄Si (M⁺) 491.2240. found491.2242 LRMS (EI) m/z 491 (M⁺), 434, 392, 376, 319, 279, 262, 223, 178,167, 149, 136, 121, 107, 91, 77, 57.

(+/−)5-Ethyl-1,4,5,13-tetrahydro-5-hydroxy-10-amino-12-trimethylsilyl-3H,15H-oxepino[3′,4′:6,7]indolizino[1,2-b]quinoline-3,15-dione(1d) 10-amino-7-trimethylsilylhomocamptothecin)

Trifluoroacetic acid (0.1 mL) was added to a solution containing CH₂Cl₂(0.5 mL) and compound (1c) (6.6 mg, 0.012 mmol) and the contents werestirred at 22° C. After 5h the mixture was poured into sat. NaHCO₃ (2mL) and extracted with EtOAc (6×2 mL). The EtOAc was dried (Na₂SO₄),concentrated and chromatographed (CH₂Cl₂:MeOH 95:5) to give (1d) 2.5 mg(45%) as an orange-red solid: ¹H NMR (300 MHz, CDCl₃/CD₃OD) δ 0.60 (s,9H), 0.94 (t, J=7 Hz, 3H), 1.92-2.05 (m, 2H), 3.16 (d, J=14 Hz, 1H),3.46 (d, J=14 Hz, 1H), 5.24 (s, 2H), 5.40 (d, J=15 Hz, 1H), 5.61 (d,J=15 Hz, 1H), 7.25-7.32 (m, 2H), 7.52 (s, 1 H), 7.89 (d, J=9 Hz, 1H);¹³C NMR (125 MHz, CDCl₃/CD₃OD) δ −0.12, 7.1, 35.7, 41.5, 51.6, 61.3,72.8, 99.3, 107.0, 120.4, 121.7, 129.9, 133.6, 134.4, 139.5, 141.0,144.7, 145.2, 146.4, 156.2, 159.3, 172.6; HRMS (EI) m/z calcd forC₂₄H₂₇N₃O₄Si (M⁺) 449.1771. found 449.1791 LRMS (EI) m/z 449 (M⁺), 434,402, 389, 374, 350, 335, 304, 178, 73. (+/−).

5-Ethyl-1,4,5-trihydro-5-hydroxy-7-iodo-8-(5-trimethylsilyl-2-pentynyl)oxepino[3,4-c]pyridine-3,9-dione(10c)

Following the procedure outlined above iodopyridone (9) (0.106 g, 0.92mmol) was N-alkylated with the 2-trimethylsilylethyl propargyl bromide(0.43 g, 1.84 mmol). Flash chromatography (CH₂Cl₂:EtOAc 10:1) gaveiodopyridone (10c) 68 mg (46%) as a light yellow foam: 1H NMR (300 MHz,CDCl₃) δ −0.069 (s, 9H), 0.72 (t, J=8 Hz, 2 H), 0.87 (t, J=7 Hz, 3H),1.70-1.88 (m, 2H), 2.10-2.20 (m, 2H), 2.96 (d, J=14 Hz, 1H), 3.15 (br s,1H), 3.27 (d, J=14 Hz, 1H), 4.90-5.00 (m, 2H), 5.07 (d, J=15 Hz, 1H),5.41 (d, J=15 Hz, 1H), 7.02 (s, 1H); ¹³C NMR (75 MHz, CDCl₃) δ −1.6,8.2, 13.5, 15.7, 35.9, 42.5, 45.3, 62.7, 72.5, 73.4, 88.0, 99.7, 119.2,122.8, 153.9, 160.4, 171.8; HRMS (EI) m/z calcd for C₁₉H₂₆INO₄Si (M⁺)487.0676. found 487.0676 LRMS (EI) m/z 487 (M⁺), 472, 400, 374, 346, 96,73.

(+/−)5-Ethyl-1,4,5,13-tetrahydro-5-hydroxy-12-(2-trimethylsilylethyl)-3H,15H-oxepino[3′,4′:6,7]indolizino[1,2-b]quinoline-3,15-dione(11) (7-(2-trimethylsilylethyl)homocamptothecin)

To an oven dried pressure tube under Ar was added the iodopyridone (10c)(16 mg, 0.033 mmol) followed by Benzene (0.5 mL). Next phenylisonitrile(10.2 mg, 0.1 mmol) and hexamethylditin (16.7 mg, 0.051 mmol) were addedand the tube was flushed with Ar, sealed and placed in front of a 275WGE sunlamp. After 12 hours of irradiation, the mixture was concentratedand chromatographed (CH₂Cl₂:acetone 4:1) to yield the desiredhomocamptothecin (11) 3.6 mg (24%) as a tan solid: ¹H NMR (300 MHz,CDCl₃) δ 0.184 (s, 9H), 0.85-1.05 (m, 5H), 1.98-2.10 (m, 2H), 3.00-3.12(m, 2H), 3.18 (d, J=14 Hz, 2H), 3.48 (d, J=13 Hz, 1H), 5.14 (d, J=19 Hz,1H), 5.24 (d, J=19 Hz, 1H), 5.34 (d, J=15 Hz, 1H), 5.71 (d, J=15 Hz,1H), 7.53 (s, 1H), 7.57-7.63 (m, 1 H), 7.70-7.77 (m, 1H), 7.94 (d, J=8Hz, 1H), 8.10 (d, J=8 Hz, 1H); HRMS (EI) m/z calcd for C₂₆H₃₀N₂O₄Si (M⁺)462.1975. found 462.1976 LRMS (EI) m/z 462 (M⁺), 447, 415, 402, 391,377, 363, 348, 317, 289, 243, 231, 73, 59.

(+/−)5-Ethyl-1,4,5,13-tetrahydro-5-hydroxy-10-(tert-butyloxycarbonylamino)-12-(2-trimethylsilylethyl)-3H,15H-oxepino[3′,4′:6,7]indolizino[1,2-b]quinoline-3,15-dione(1j)(10-tert-butyloxycarbonylamino-7-(2-trimethylsilylethyl)homocamptothecin)

Following the procedure described above, iodopyridone (10c) (16 mg,0.033 mmol) was reacted with para-Bocaminophenylisonitrile (21.6 mg, 0.1mmol) and hexamethylditin (16.7 mg, 0.051 mmol) in benzene (0.5 mL).Chromatography (CH₂Cl₂:Acetone 7:1) gave compound (1j) 10.7 mg (56%) asa brown solid: ¹H NMR (300 MHz, CDCl₃) δ 0.20 (s, 9H), 0.83-0.93 (m,2H), 0.99 (t, J=7 Hz, 3H), 1.60 (s, 9H), 1.93-2.10 (m, 2H), 2.90-3.05(m, 2H), 3.24 (d, J=14 Hz, 1H), 3.44 (d, J=14 Hz, 1H), 3.74 (br s, 1H),5.03 (d, J=19 Hz, 1H), 5.20 (d, J=19 Hz, 1H), 5.33 (d, J=15 Hz, 1H),5.67 (d, J=15 Hz, 1H), 6.85 (s, 1H), 7.35-7.44 (m, 2H), 7.85 (d, J=9 Hz,1H), 8.11 (br s, 1 H); HRMS (EI) m/z calcd for C₃₁H₃₉N₃O₆Si (M⁺)577.2608. found 577.2611 LRMS (EI) m/z 577 (M⁺), 521, 477, 462, 434,417, 378, 304, 260, 178, 108, 73.

(+/−)5-Ethyl-1,4,5,13-tetrahydro-5-hydroxy-10-acetoxy-12-(2-trimethylsilylethyl)-3H,15H-oxepino[3′,4′:6,7]indolizino[1,2-b]quinoline-3,15-dione(1k) (10-acetoxy-7-(2-trimethylsilylethyl)homocamptothecin)

Following the procedure described above, iodopyridone (10c) (16 mg,0.033 mmol) was reacted with para-acetoxyphenylisonitrile (16 mg, 0.1mmol) and hexamethylditin (16.7 mg, 0.051 mmol) in benzene (0.5 mL).Chromatography (CH₂Cl₂:Acetone 5:1) gave compound (1k) 7.1 mg (41%) as atan solid: ¹H NMR (300 MHz, CDCl₃) δ 0.19 (s, 9H), 0.82-0.89 (m, 2H),0.99 (t, J=7 Hz, 3H), 1.95-2.06 (m, 2H), 2.42 (s, 3H), 2.94-2.98 (m,2H), 3.23 (d, J=14 Hz, 1H), 3.46 (d, J=14 Hz, 1H), 3.59 (br s, 1H), 5.08(d, J=19 Hz, 1H), 5.24 (d, J=19 Hz, 1H), 5.35 (d, J=15 Hz, 1H), 5.68 (d,J=15 Hz, 1H), 7.41-7.49 (m, 2H), 7.60 (d, J=2 Hz, 1H), 7.96 (d, J=9 Hz,1H); HRMS (EI) m/z calcd for C₂₈H₃₂N₂O₆Si (M⁺) 520.2030. found 520.2017LRMS (EI) m/z 520 (M⁺), 491, 478, 463, 449, 431, 421, 406, 393, 379,333, 305, 261, 178, 109, 73.

(+/−)5-Ethyl-1,4,5,13-tetrahydro-5-hydroxy-10-hydroxy-12-(2-trimethylsilylethyl)-3H,15H-oxepino[3′,4′:6,7]indolizino[1,2-b]quinoline-3,15-dione(11) (10-hydroxy-7-(2-trimethylsilylethyl)homocamptothecin)

Following the procedure outlined above, compound (1k) (7.1 mg, 0.014mmol) was reacted with K₂CO₃ (4 mg, 0.028 mmol) in a MeOH/H₂O solution.The residue was chromatographed (CH₂Cl₂:Acetone 7:1) to give compound(II) 2.6 mg (39%) as a yellow solid: ¹H NMR (300 MHz, CDCl₃/CD₃OD) δ0.042 (s, 9H), 0.68-0.92 (m, 5H), 1.80-1.95 (m, 2H), 2.83-2.95 (m, 2H),3.07 (d, J=14 Hz, 1H), 3.28 (d, J=14 Hz, 1H), 5.05 (s, 2H), 5.29 (d,J=15 Hz, 1H), 5.51 (d, J=15 Hz, 1H), 7.22 (d, J=2 Hz, 1H), 7.26-7.34 (m,1H), 7.40 (s, 1H), 7.89 (d, J=9 Hz, 1H); HRMS (EI) m/z calcd forC20H₃₀N2O₅Si (M⁺) 478.1924. found 478.1915 LRMS (EI) m/z 478 (M⁺), 463,431, 418, 393, 379, 364, 305, 261, 153, 117, 105, 91, 73, 59.

(+/−)5-Ethyl-1,4,5,13-tetrahydro-5-hydroxy-10-amino-12-(2-trimethylsilylethyl)-3H,15H-oxepino[3′,4′:6,7]indolizino[1,2-b]quinoline-3,15-dione(1m) (10-amino-7-(2-trimethylsilylethyl)homocamptothecin)

Trifluoroaceticacid (0.1 mL) was added to a solution containing CH₂Cl₂(0.5 mL) and compound (II) (10.7 mg, 0.018 mmol) and the contents werestirred at 22° C. After 5 h, the mixture was poured into sat. NaHCO₃ (2mL) and extracted with EtOAc (6×2 mL). The EtOAc was dried (Na₂SO₄),concentrated and chromatographed (CH₂Cl₂:MeOH 96:4) to give (1m) 6.7 mg(78%) as a yellow solid: ¹H NMR (300 MHz, CDCl₃/CD₃OD) δ 0.059 (s, 9H),0.70-0.92 (m, 5H), 1.82-1.98 (m, 2H), 2.80-2.92 (m, 2H), 3.08 (d, J=14Hz, 1H), 3.29 (d, J=14 Hz, 1H), 5.00 (s, 2H), 5.29 (d, J=15 Hz, 1H),5.52 (d, J=15 Hz, 1H), 6.95 (d, J=2 Hz, 1H), 7.18 (dd, J₁=9 Hz, J₂=2 Hz,1H), 7.38 (s, 1H), 7.83 (d, J=9 Hz, 1H).

Although the present invention has been described in detail inconnection with the above examples, it is to be understood that suchdetail is solely for that purpose and that variations can be made bythose skilled in the art without departing from the spirit of theinvention except as it may be limited by the following claims.

1. A compound having the formula

in racemic form, enantiomerically enriched form or enantiomerically pureform; wherein R¹ and R² are independently the same or different and arehydrogen, —C(O)R^(f) wherein R^(f) is an alkyl group, an alkoxy group,an amino group or a hydroxy group, an alkyl group, an alkenyl group, analkynyl group, an alkoxy group, an aryloxy group, an acyloxy group,—OC(O)OR^(d), wherein R^(d) is an alkyl group, —OC(O)NR^(a)R^(b) whereinR^(a) and R^(b) are independently the same or different, H, —C(O)R^(f),an alkyl group or an aryl group, a halogen, a hydroxy group, a nitrogroup, a cyano group, an azido group, a formyl group, a hydrazino group,an amino group, —SR^(c), wherein R^(c) is hydrogen, —C(O)R^(f), an alkylgroup or an aryl group; or R¹ and R² together form a chain of three orfour members selected from the group of CH, CH₂, O, S, NH, or NR¹⁵,wherein R¹⁵ is an C₁-C₆ alkyl group; R³ is H, a halogen atom, a nitrogroup, an amino group, a hydroxy group, or a cyano group; or R² and R³together form a chain of three or four members selected from the groupof CH, CH₂, O, S, NH, or NR¹⁵, wherein R¹⁵ is an C₁-C₆ alkyl group; R⁴is H, F, an amino group, a C₁₋₃ alkyl group, a C₂₋₃ alkenyl group, aC₂₋₃ alkynyl group, a trialkylsilyl group or a C₁₋₃ alkoxy group; R⁵ isa C₁₋₁₀ alkyl group, an alkenyl group, an alkynyl group, or a benzylgroup; R⁶ is —Si (R⁸R⁹R¹⁰) or —(R⁷)Si(R⁸R⁹R¹⁰), wherein R⁷ is analkylene group, an alkenylene group, or an alkynylene group; and R⁸, R⁹and R¹⁰ are independently a C₁₋₁₀ alkyl group, a C₂₋₁₀ alkenyl group, aC₂₋₁₀ alkynyl group, an aryl group or a —(CH₂)_(N)R¹¹ group, wherein Nis an integer within the range of 1 through 10 and R¹¹ is a hydroxygroup, an alkoxy group, an amino group, an alkylamino group, adialkylamino group, a halogen atom, a cyano group, —SR^(c) or a nitrogroup; R¹² is H or —C(O)R^(f), —C(O)OR^(d) or —C(O)NR^(a)R^(b); R¹³ isH, F or —CH₃; and pharmaceutically acceptable salts thereof.
 2. Thecompound of claim 1 wherein R² and R³ together form a group of theformula —O(CH₂)_(n)O— wherein n represents the integer 1 or
 2. 3. Thecompound of claim 1 wherein R⁵ is an ethyl group, an allyl group, abenzyl group or a propargyl group.
 4. The compound of claim 1 whereinR¹³ is H.
 5. The compound of claim 4 wherein R⁵ is an ethyl group. 6.The compound of claim 5 wherein R⁴ is H.
 7. The compound of claim 6wherein R⁸ and R⁹ are methyl groups, R¹⁰ is a tert-butyl group or amethyl group, R¹ is H and R³ is H.
 8. The compound of claim 7 wherein R²is H, NH₂ or OH.
 9. A compound having the formula

wherein R⁵ is a C₁₋₁₀ alkyl group, an alkenyl group, an alkynyl group,or a benzyl group; R¹² is H or —C(O)R^(f), —C(O)OR^(d) or—C(O)NR^(a)R^(b).
 10. The compound of claim 9 wherein R⁵ is an ethylgroup, an allyl group, a benzyl group or a propargyl group.
 11. Thecompound of claim 10 wherein R⁵ is an ethyl group.
 12. A compound havingthe formula

wherein R⁵ is a C₁₋₁₀ alkyl group, an alkenyl group, an alkynyl group,or a benzyl group; R¹³ is H, F or —CH₃; and R¹⁵ is a C₁-C₆ alkyl group.13. The compound of claim 12 wherein R⁵ is an ethyl group, an allylgroup, a benzyl group or a propargyl group.
 14. The compound of claim 13wherein R¹³ is H.
 15. The compound of claim 14 wherein R⁵ is an ethylgroup.
 16. A compound having the formula

in racemic form, enantiomerically enriched form or enantiomerically pureform; wherein X is a radical precursor; R⁵ is a C₁₋₁₀ alkyl group, analkenyl group, an alkynyl group, or a benzyl group; R⁶ is an alkylgroup, —Si(R⁸R⁹R¹⁰) or —(R⁷) Si(R⁸R⁹R¹⁰), wherein R⁷ is an alkylenegroup, an alkenylene group, or an alkynylene group; and R⁸, R⁹ and R¹⁰are independently a C₁₋₁₀ alkyl group, a C₂₋₁₀ alkenyl group, a C₂₋₁₀alkynyl group, an aryl group or a —(CH₂)_(N)R¹¹ group, wherein N is aninteger within the range of 1 through 10 and R¹¹ is a hydroxy group,alkoxy group, an amino group, an alkylamino group, a dialkylamino group,a halogen atom, a cyano group, —SR^(c) or a nitro group; and R¹³ is H, For —CH₃.
 17. The compound of claim 16 wherein R⁵ is an ethyl group, anallyl group, a benzyl group or a propargyl group.
 18. The compound ofclaim 17 wherein X is Br or I.
 19. The compound of claim 18 wherein R¹³is H.
 20. The compound of claim 19 wherein R⁵ is an ethyl group.